Method for preparing a stable TNF-α vaccine

ABSTRACT

A stable immunogenic product for the induction of antibodies against one or more antigenic proteins in a subject, characterized in that it comprises proteinaceous immunogenic heterocomplexes which are formed by associations between (i) antigenic protein molecules and (ii) proteinaceous carrier molecules and in that less than 40% of the antigenic proteins (i) are linked to the proteinaceous carrier molecules (ii) by a covalent bond.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a continuation-in-part of applicationSer. No. 10/527,975, filed Mar. 15, 2005 now U.S. Pat. No. 7,972,603,which is a nationalization of International application No.PCT/FR03/002733, filed Sep. 16, 2003, and published in French, both ofwhich are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to stable immunogenic products comprisingimmunogenic protein heterocomplexes for obtaining a humoral immuneresponse with production of specific antibodies raised against one oremore antigens, in particular against a “self” antigen, as well as theiruse in the field of vaccines.

2. Related Art

Obtaining a high level antibody response from a given antibody, in anindividual, is an object commonly sought, whether the antigen is a“foreign” antigen or a “self” antigen.

However, the problem of a good recognition of the antigen against whichan antibody response is being sought, in an individual, should be solvedin a number of cases, more particularly (a) when the antigen of interestbehaves like a “hapten”, i.e. a low molecular mass chemical structurewhich is little or not immunogenic under a free form, but that, oncefixed on a high molecular mass molecule, is able to induce theproduction of specific antibodies of such a hapten, and (b) when theantigen of interest is a self protein, i.e. a protein being naturallyproduced in the individual, for which there exists an immune tolerancedue to the deletion of corresponding lymphocyte T clones, during thedevelopment of the immune system.

In order to cause, or increase, the recognition of an antigen ofinterest by B cells, various immunogenic constructions were developed inthe state of the art.

A first immunogenic construction form comprises a covalent coupling ofthe antigen of interest on a carrier molecule, the carrier moleculebringing structures recognized by the auxiliary T lymphocytes (“Thelper” cells), in association with class II molecules of theHistocompatibility Major Complex (HMC), and activating the auxiliary Tlymphocytes then producing various cytokins, amongst which IL-2, saidcytokins activating in turn the specific B cell clones of the antigen ofinterest. The specific B cells of the antigen of interest, onceactivated, multiply and produce antibodies specific to the antigen ofinterest, which is the objective being sought. Generally, such a type ofimmunogenic constructions comprises products of the covalent chemicalcoupling between the antigen of interest and the carrier molecule,which, after purification and removal steps of the non coupled products,are final products with a well defined chemical structure.

The first form of an immunogenic construction is for example illustratedby the article by Richard and al. describing the preparation of productsof the covalent coupling between IL-9 and ovalbumin (Proc. Natl. Acad.Sci. USA, Vol. 97(2): 767-772). It is also illustrated in such U.S. Pat.No. 6,340,461 (Terman) which discloses coupling products between one ormore copies of an antigen of interest, against which a specific antibodyresponse is being sought in an individual, and a carrier moleculeconsisting in a “Superantigen”. The antigen of interest is coupledexclusively covalently to the carrier molecule, for example, by means ofglutaraldehyde (also called “pentanedial”), the non-covalently coupledproducts being removed in order to obtain a chemically well-definedfinal product.

Optionally, the product from the covalent coupling between the antigenof interest and the superantigen could be prepared in the form of apolymer of said coupling product, for example, through a non covalentbond of the monomeric coupling products between one another, throughionic interactions, adsorption interactions as well as biospecificinteractions. For example, the monomeric coupling products could formcomplexes with highly positively or negatively charged molecules,through salt bridges produced in low ionic strength conditions. Largescomplexes of monomeric coupling products are prepared using chargedpolymers such as poly(L-glutamic acid) or poly(L-lysine) polymers.According to another embodiment of a monomeric coupling product polymer,the exclusively covalent coupling products between the antigen ofinterest and the superantigen could be adsorbed or coupled noncovalently at the surface of microparticles, such as latex beads orother hydrophobic polymers.

A second embodiment of such immunogenic constructions commonly called“MAP” structure (for “Multi-Antigenic Protein”) generally have the formof a protein backbone comprised of a linear or branched, poly(lysine)polymer, onto which one or more antigens of interest are covalentlybound.

A third embodiment of such immunogenic constructions consists inmicroparticles onto which fixed the antigen(s) of interest is/are bound.Various forms of antigen carrier microparticles are known.

For example, iscomes (for “immunostimulating complexes”) are knowncomprised of an antigenic complex and an adjuvant, the QuilA compound.

Liposomes are also known having the same drawbacks as the iscomes, i.e.more particularly some toxicity and immunological side effects, due totheir lack of purity.

Biodegradable microparticles are also known such as lactic acid andglutamic acid polymers (Aguado and Lambert, 1992, Immuno. Biol., Vol.184: 113-125) as well as starch particles (U.S. Patent Application2002/0098203—Gutavsson et al.), in the polymeric matrix of whichantigens of interest are trapped. Such particles release the antigenunder their soluble form during the degradation of the polymeric matrix.

Particles have also been disclosed exclusively comprised of hybridrecombinant proteins, as disclosed in French Patent Application FR2,635,532 (Thiollais et al.).

Porous microspheres are also known wherein the antigens are immobilizedwithin micropores through captation or physical coupling, as disclosedin the U.S. Pat. No. 5,008,116 (Cahn).

However, the various solutions suggested in the state of the art sharein common at least one technical inconvenient related to theirpreparation method, i.e. the loss of a high proportion of the antigenicmaterial of interest, due to a necessary step for removing the noncoupled or non adsorbed antigens.

Moreover, while the prior art techniques allow to provide an associationbetween the low molecular mass antigen of interest with a carriermolecule, they are generally not adapted to coupling a high molecularmass antigen of interest, for example, of more than 10 kDa, with thecarrier molecule, because, in particular, of steric hindrancespreventing coupling a high number of molecules of antigens of interesthaving a high molecular mass with an identical carrier molecule.

Finally, most if not all the known peptide antigenic constructionsencompass in their structure a single carrier molecule, which is atechnical inconvenient when the objective is to induce a preventive ortherapeutic immune response both against the antigen of interest and thecarrier molecule itself.

There is therefore a need in the state of the art for improvedimmunogenic constructions allowing for the production of a high level ofantibodies specific to an antigen of interest in an individual wheresuch a humoral immune response is sought, being less expensive, simpleto prepare and able to be synthesized reproducibly.

SUMMARY OF THE INVENTION

The present invention provides new immunogenic constructions allowingthe solution of various technical problems encountered with theimmunogenic constructions as known in the prior art and allowing themeeting of the above-described various technical needs.

The object of the invention is to provide a stable immunogenic productfor inducing antibodies raised against one or more antigenic proteins ina subject, characterized in that it comprises protein immunogenicheterocomplexes consisting of associations between (i) antigenic proteinmolecules and (ii) carrier protein molecules and in that less than 40%of the antigenic proteins (i) are covalently linked to carrier proteinmolecules (ii).

Another object of the invention is also an immunogenic productcomprising stable protein immunogenic heterocomplexes for inducingantibodies raised against one or more antigenic proteins in a subject,each heterocomplex comprising (i) a plurality of antigenic proteins,linked to a (ii) carrier protein molecule, characterized in that lessthan 40% of the antigenic proteins (i) are covalently linked to carrierprotein molecules (ii).

Preferably, the immunogenic heterocomplex making up the immunogenicproduct according to the invention comprises 5 to 50 antigenic proteins(i) for one carrier protein molecule (ii), preferably 20 to 40 antigenicproteins (i) for one carrier protein molecule (ii).

Preferably, the covalent bonds between one or more antigenic proteins(i) and the carrier protein molecules (ii) occur by means of afunctional binding chemical agent.

It is meant under antigenic molecule of interest, any protein comprisingone or more B epitopes of a native antigenic protein against which theproduction of antibodies is being sought. Said antigenic molecule ofinterest can consist in the native protein itself or a protein derivateof the native protein, such as a peptide fragment of the native protein,as well as any biologically inactivated form of the native proteinobtained through chemical, physical treatment or genetic mutation. Theantigenic molecule of interest could also consist in a homo-oligomer ora homo-polymer of the native protein as well as a homo-oligomer or ahomo-polymer of a peptide fragment of the native protein. The antigenicprotein of interest could also consist in a hetero-oligomer or ahetero-polymer comprising a combination of several distinct peptidefragments initially included in the native protein.

According to the general embodiment of an immunogenic product accordingto the invention, the carrier protein molecule (ii) is an immunogenicprotein inducing the production of T helper lymphocytes and/or ofcytotoxic T lymphocytes raised against cells having at their surfacesaid carrier protein molecule or any peptide being derived therefrom, inassociation with presenting molecules of the Major HistocompatibilityComplex (MHC), respectively of class I and/or class II. The carrierprotein molecule (ii) could also be an immunogenic protein inducing boththe production of T helper lymphocytes and the production of antibodiesby B lymphocytes raised against the carrier protein.

According to an embodiment of a particular interest, the immunogenicproduct is characterized in that the carrier protein molecule (ii) is animmunogenic protein inducing the production of T cytotoxic lymphocytesraised against cells having at their surface said carrier proteinmolecule or any peptide being derived therefrom, in association withmolecules of the Major Histocompatibility Complex (MHC) class I.

The preferred immunogenic products according to the invention areselected amongst immunogenic products comprising the followingheterocomplexes, wherein the antigenic proteins (i), on the one hand,and the protein carrier molecule (ii), on the other hand, arerespectively:

a) (i) IL-4 and (ii) KLH;

b) (i) alpha interferon and (ii) KLH;

c) (i) VEGF and (ii) KLH;

d) (i) IL-10 and (ii) KLH;

e) (i) alpha interferon and (ii) gp 160 of VIH1

f) (i) IL-4 and (ii) the Bet v 1 allergenic antigen; and

g) (i) VEGF and (ii) the papillomavirus E7 protein;

h) (i) the inactivated VIH1 Tat protein and (ii) the VIH1 gp120 protein.

i) (i) an IgE isotype human antibody and (ii) the inactivated VIH1 Tatprotein;

j) (i) the ricin β fragment and (ii) KLH.

The invention also relates to a composition, more particularly, apharmaceutical composition, an immunogenic composition or a vaccinecomposition, characterized in that it comprises an immunogenic productsuch as hereinabove described.

It also relates to a method for preparing an immunogenic productaccording to any one of claims 1 to 16, characterized in that itcomprises the following steps of:

a) incubating the antigenic proteins (i) and the carrier molecule (ii)in a molar ratio (i):(ii) ranging from 10:1 to 50:1 in the presence of abinding chemical; and

b) collecting the immunogenic product comprising immunogenicheterocomplexes being prepared in step a).

Other objects, features, and advantages of the present invention will beapparent to those skilled in the art upon a reading of thisspecification including the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is better understood by reading the following DetailedDescription of the Preferred Embodiments with reference to theaccompanying drawing figures, in which like reference numerals refer tolike elements throughout, and in which:

FIG. 1 illustrates the characterization of the immunogenic productcomprising murine KLH-VEGF heterocomplexes through isoelectrofocusing inan agarose gel followed by the emergence of proteins throughimmuno-blotting (“Western Blot”).

FIG. 2 illustrates the characterization of the immunogenic productcomprising human KLH-VEGF heterocomplexes through isoelectrofocusingthrough a coloration with Coomassie blue, followed by an immunoblotting(“Western Blot”). The isoelectrofocusing gel is represented at the leftof the figure. The immunoblotting gels using anti-KLH (left) or humananti-VEGF (right) antibodies are illustrated on the right of the figure.

FIG. 3 illustrates the characterization of the immunogenic productcomprising human KLH-IL4 heterocomplexes through isoelectrofocusing inan agarose gel followed by the emergence of proteins throughimmunoblotting (“Western Blot”).

FIG. 4 illustrates the characterization of the immunogenic productcomprising gp 160-IFNα complexes through isoelectrofocusing in anagarose gel followed by the mergence of proteins through immunoblotting(“Western Blot”).

FIG. 5 illustrates the immunogenic (humoral) activity of the murineKLH-VEGF immunogenic product through determination of the title antibodyobtained after an immunization of mice. FIG. 5A relates to miceimmunized with murine VEGF. FIG. 5B relates to mice immunized with theimmunogenic product comprising KLK-VEGF heterocomplexes. FIG. 5Cillustrates control mice injected with Freund's Incomplete Adjuvant(FIA).

FIG. 6 shows the immunogenic (humoral) activity of the murine KLH-VEGFimmunogenic product, through determination of the neutralizing power ofantibodies obtained after immunization, towards the angiogenic activityof the VEGF protein.

FIG. 7 illustrates the immunogenic (humoral) activity of the humanKLH-VEGF immunogenic product, through determination of the antibodytitle obtained after immunization of mice.

FIG. 8 illustrates the immunogenic (humoral) activity of the humanKLH-VEGF immunogenic product, through determination of the neutralizingpower of antibodies obtained after immunization, towards the angiogenicactivity of the VEGF protein, measured through the proliferation ofendothelial cells.

FIG. 9 illustrates the immunogenic (humoral) activity of the murineKLH-IL4 immunogenic product, through determination of the title antibodyobtained after immunization.

FIG. 10 illustrates the immunogenic (humoral) activity of the murineKLH-IL4 immunogenic product, through determination of the neutralizingpower of antibodies obtained after immunization, towards the inducingactivity of the proliferation of HT-2 cells by the IL4.

FIG. 11 illustrates the results of the production of the IgG and IgEclass antibodies raised against Bet v 1, after the injection ofbirch-tree pollen, to mice preliminarily immunized with an immunogenicproduct according to the invention comprising KLH-IL4 complexes.

FIG. 12 illustrates the immunogenic (humoral) activity of the humanKLH-IL4 immunogenic product through determination of the neutralizingpower of antibodies obtained after immunization, towards the inducingactivity of the proliferation of HT-2 cells by the ILA.

FIG. 13 consists of a scheme that illustrates the general procedure formanufacturing a stable immunogenic product according to the invention.

FIGS. 14A and 14B illustrate the humoral response in mice immunizedrespectively with (A) KLH alone or (B) a stable immunogenic productmanufactured as disclosed in Example 35. Abscissa: individuals;Ordinate: IgG anti-TNFa antibody titers.

FIGS. 15A and B illustrate the anti-TNFa neutralizing activity of theserum antibodies produced by mice immunized respectively by (A) KLHalone or (B) a stable immunogenic product manufactured as disclosed inExample 35. Each curve corresponds to the blood serum form one mouse.Abscissa: percent neutralization of the TNFa activity; Ordinate: serumdilution.

FIGS. 16A, 16B, and 16C illustrate the humoral response of Rhesusmacaques immunized respectively with (A) KLH alone, (B) 20 μg of) astable immunogenic product manufactured as disclosed in Example 35, and(C) 80 μg of a stable immunogenic product manufactured as disclosed inExample 35. Abscissa: Days after first injection; Ordinate: IgGanti-TNFa antibody titers.

FIGS. 17A, 17B, and 17C illustrates the anti-TNFa neutralizing activityof the serum antibodies produced by Rhesus macaques immunizedrespectively with (A) KLH alone, (B) 20 μg of a stable immunogenicproduct manufactured as disclosed in Example 35, and (C) 80 μg of astable immunogenic product manufactured as disclosed in Example 35.Abscissa: percent neutralization of the TNFa activity; Ordinate: serumdilution.

FIGS. 18A, 18B, and 18C illustrate the humoral response of transgenichuTNFa B6.SJL-Tg(TNF) N2 mice immunized respectively with (A) ControlPBS buffer, (B) KLH alone, (C) of a stable immunogenic productmanufactured as disclosed in Example 35 and (D) of a stable immunogenicproduct manufactured as disclosed in Example 35 combined withmethotrexate. Abscissa: individuals; Ordinate: IgG anti-TNFa antibodytiters.

FIG. 19 illustrates the arthritis clinical scores determined intransgenic huTNFa B6.SJL-Tg(TNF) N2 injected with

-   -   NaCl [♦];    -   KLH alone [▪];    -   a stable immunogenic product manufactured as disclosed in        Example 35 [▴]; and    -   a stable immunogenic product manufactured as disclosed in        Example 1 combined with methotrexate [▪]

Abscissa: Days after first injection; Ordinate: arthritis clinicalscores.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In describing preferred embodiments of the present invention illustratedin the drawings, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected, and it is to be understood that eachspecific element includes all technical equivalents that operate in asimilar manner to accomplish a similar purpose.

The invention provides new immunogenic constructions inducing a highlevel of production of antibodies specific to an antigen of interest, inan individual.

The Immunogenic Protein Heterocomplexes According to the Invention

It has been shown according to the invention that the production of ahigh level of antibodies specific to an antigen of interest could beobtained, in an individual, through the immunization of such anindividual with an immunogenic product where said antigen of interest isassociated with a carrier protein molecule, the association between saidantigen of interest and said carrier protein being partially covalentand partially non covalent.

More specifically, it has been shown according to the invention that anexcellent antibody response raised against an antigen of interest isobtained when an individual is being immunized with a stable immunogenicproduct comprising protein heterocomplexes, wherein the heterocomplexescomprise stable associations between antigen of interest and saidcarrier protein molecule and wherein only a low proportion of suchassociations is due to a covalent bond between the antigen of interestand the carrier protein molecule, the other associations between theantigen of interest and the carrier protein molecule being produced byweak bonds, ionic interactions, hydrogen bonds, Van der Waals forces,etc.

In particular, it has been shown according to the invention that anoptimum antibody response is reached when, in a stable immunogenicproduct such as described hereinabove, less than 40% of the molecules ofthe antigen of interest are covalently linked to the carrier proteinmolecules. According to the invention, an antigenic molecule of interestis covalently linked to a carrier protein molecule by “one” covalentbond means that said molecule of antigen of interest is covalentlylinked, chemically, to said carrier protein molecule, by at least onecovalent bond, i.e. optionally by two covalent bonds or more.

The percentage of carrier protein molecules and of antigenic proteinproteins of interest linked between one another through covalent bondsin an immunogenic product of the invention can be easily checked by theman of the art.

For example, determining the percentage of antigenic molecules ofinterest linked to the carrier protein molecules through a covalent linkin an immunogenic product of the invention could be made using thefollowing steps of:

(i) submitting said immunogenic product in solution to denaturing andreducing conditions;

(ii) performing a size exclusion chromatography step with the product asobtained at the end of step (ii) during which the various proteincomponents with decreasing molecular mass are successively eluted fromthe size exclusion chromatography support;

(iii) measuring the amount of antigen of interest linked through acovalent bond to the carrier molecule in the eluate fraction containingthe protein components with the highest molecular mass;

(iv) comparing the amount of antigen of interest measured in step (iii)with the total amount of antigen of interest initially included in thestarting immunogenic product.

In step (i) of the method for determining the above-described covalentlink percentage, incubating a given amount (in number of moles or inweight) of the immunogenic product of the invention under denaturing andreducing conditions leads to a disassociation of the weak bonds betweenthe various protein components not linked between one another through acovalent bond.

Amongst preferred denaturing conditions there is the presence of urea,for example, in the final 8M concentration, or the presence of SDS, forexample, in the 1% final concentration in total weight of the solutioncontaining the immunogenic product. Amongst preferred reducingconditions there is the presence of β-mercaptoethanol, for example inthe 5% final concentration of the total volume of the solutioncontaining the immunogenic product.

In step (ii) of the method for determining the percentage of moleculesof antigen of interest and molecule of carrier protein linked betweenone another through covalent bonds, the size exclusion chromatographysupport is selected by the man of the art according to his technicalgeneral knowledge. For example, the man of the art could make use ofchromatographic supports as marketed by the Pharmacia Corporation underthe “Superdex 75™” and “Superdex 200™” trademarks.

In step (ii), the molecular fraction corresponding to the carriermolecule covalently linked to the molecules of antigen of interest iseluted first, before the eluate fraction(s) containing the antigen ofinterest under a free form. The antigen of interest being eluted under afree form corresponds to the fraction of the antigen of interest, whichwas not covalently linked to the carrier molecule, within the startingimmunogenic product. It is on the high molecular mass protein fractionthat occurs the measurement of the amount of the antigen of interestcovalently linked to the carrier protein molecule, for example, in animmuno-enzyme test, in a radioimmunologic test or in animmunofluorescence test, either direct or indirect (“sandwich”), usingantibodies specific to the antigen of interest and which do not have anyimmunologic reaction crossed with the carrier protein molecule.

In step (iii), the amount of the antigen of interest covalently linkedwith the carrier protein molecule, being measured as describedhereinabove, is compared with the initial amount of the antigen ofinterest being included in the given amount (in number of moles or inweight) of the starting immunogenic product and the percentage of theantigen of interest is thereby calculated, which is covalently linked tothe carrier protein molecule, in the immunogenic product of theinvention.

The percentage of carrier protein molecules and of antigenic proteinproteins of interest linked between one another through covalent bonds,in an immunogenic product of the invention, can be easily checked by theman of the art, making use of a second method comprising the followingsteps of:

a) immobilizing on a support of specifically antibodies raised againstthe carrier protein;

b) bringing into contact the antibodies raised against the carrierprotein, which were immobilized on the support in step a), with a knownamount of molecules of the immunogenic product to be tested comprisingsaid carrier protein and an antigenic protein of interest;

c) removing the molecules of the immunogenic product which are notlinked to the anti-carrier protein antibodies immobilized in step a), bymeans of a buffering aqueous solution comprising one ore more proteindenaturing agents;

d) d1) bringing into contact (i) immunogenic complexes formed in step c)between the immobilized anti-carrier protein antibodies and themolecules of the immunogenic product with (ii) antibodies specificallyraised against the carrier protein;

-   -   d2) separately from step d1), bringing into contact the        immunogenic complexes formed in step c) between the immobilized        anti-carrying protein antibodies and the molecules of the        immunogenic product with (ii) antibodies specifically raised        against the antigenic protein of interest;

e) e1) quantifying the antibodies added in step d1) having been linkedto the carrier protein;

-   -   e2) quantifying the antibodies added in step d2) having been        linked to the antigenic protein;

f) calculating the ratio between:

(i) the amount of anti-carrier protein bound antibodies measured in stepe1); and

(ii) the amount of anti-carrier protein bound antibodies measured instep e2),

said ratio consisting in the proportion of carrier protein molecules andantigenic protein molecules of interest being linked between one anotherthrough covalent bonds, within the starting immunogenic product.

In step c) of the above described method, the use of an aqueous washingsolution containing one or more protein denaturing agents leads to adenaturation of the immunogenic product linked to the anti-carrierprotein antibodies, resulting in the release, in the washing solution,of antigenic protein molecules of interest which are not covalentlylinked to the carrier protein molecules. Therefore, in step d2) of themethod, only the antigenic protein molecules of interest beingcovalently linked to the carrier protein molecules are quantified.

Preferably, the denaturing buffering solution used in step c) contains asurfactant such as Tween®20, in a final concentration of 0.1% v/v.

in steps d1) and d2), the amounts of bound antibodies are preferablymeasured through incubating antigen-antibodies complexes formed at theend of each of said steps with a new antibody being labeled through adetectable molecule, respectively:

(i) in step d1), a new antibody directed against an the anti-carrierprotein antibody and labeled with a detectable molecule;

(ii) in step d2), a new antibody directed against an antibodyanti-antigenic protein of interest and labeled with a detectablemolecule.

The detectable molecule is indiscriminately either a radioactivemolecule, a fluorescent molecule or an enzyme. As an enzyme, peroxydasecould more particularly be used, its presence being revealed throughcolorimetry, after incubation with the ortho-phenylenediamine (OPD)substrate.

A detailed protocol of the above-mentioned method is described in theexamples.

By way of illustration, it has been shown according to the invention,using the first or the second above described quantification methodsthat:

-   -   in the immunogenic product comprising heterocomplexes between        the KLH carrier molecule and human alpha interferon molecules,        from 3 to 8% of the alpha interferon molecules are covalently        linked to the KLH carrier protein molecule;    -   in the immunogenic product comprising the heterocomplexes        between the KLH carrier protein molecule and murine IL-4        molecules, about 11% of the IL-4 molecules are covalently linked        to the KLH carrier protein molecule.

Obviously, depending on the preparations, the percentage of molecules ofantigenic protein of interest covalently linked to the carrier proteinmolecules could significantly vary. However, in all cases, such apercentage is always lower than 40%.

The object of the invention is to provide a stable immunogenic productfor inducing antibodies raised against one or more antigenic proteins ina subject, characterized in that it comprises protein immunogenicheterocomplexes comprising associations between (i) antigenic proteinmolecules and (ii) carrier protein molecules and in that less than 40%of the antigenic proteins (i) are covalently linked to carrier proteinmolecules (ii).

Another object of the invention is also to provide an immunogenicproduct comprising stable protein immunogenic heterocomplexes forinducing antibodies raised against one or more antigenic proteins in asubject, each heterocomplex comprising (i) a plurality of antigenicproteins, linked to a (ii) carrier protein molecule, characterized inthat less than 40% of the antigenic proteins (i) are covalently linkedto carrier protein molecules (ii).

Most preferably, the antibodies with their production being induced bythe immunogenic product of the invention comprise “neutralizing” or“blocking” antibodies. A “neutralizing” or a “blocking” antibody isdefined, according to the invention, as an antibody the binding of whichon the native protein blocks the biological activity of such a nativeprotein, which is an important objective being sought by the invention,when the native protein against which the antibodies are raised has adeleterious biological activity for the organism, within the targetedpathological context of an individual to be treated, for example, whenthe native protein has an angiogenic activity, an immunosuppressiveactivity, as well as an allergenic activity, more particularly aninterleukin-4 production inducing activity.

A “carrier protein molecule”, included in the immunogenic product of theinvention, means any protein or peptide being at least 15 amino acidslong, whatever its amino acid sequence, and which, when partiallycovalently being associated to the molecules of the antigen of interestfor forming protein heterocomplexes making up the immunogenic product ofthe invention, allows for a large number of molecules of the antigen ofinterest to be presented to the B lymphocytes.

According to a first aspect, the carrier protein molecule consists inone protein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising one or more auxiliary T epitopes(“helper”) able to activate auxiliary T lymphocytes (“T helper”) of thehost organism for producing cytokins, including interleukin 2, suchcytokins, in turn, activating and inducing the proliferation of Blymphocytes, which, after maturation, will produce antibodies raisedagainst the antigenic protein (i).

According to a second aspect, a carrier protein molecule consists in oneprotein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”), as described in the above-mentioned first aspect,one or more cytotoxic T epitopes, able to induce a cell immune responsethrough the production of cytotoxic T lymphocytes specific of thecarrier protein molecule, such lymphocytes being able to specificallyrecognize cells expressing on their surface said carrier protein or anypeptide being derived therefrom, in association with class 1Histocompatibility Major Complex (HMC) molecules. If need be, thecarrier protein molecule consists in one oligomer of one protein or onepeptide, further comprising besides one or more T helper epitopes, oneor more above defined cytotoxic T epitopes.

According to a third aspect, a carrier protein molecule consists in oneprotein or one peptide being at least 15 amino acid long, as well as oneoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”) as defined in the first aspect, one or more Bepitopes, able to induce the production of antibodies by lymphocytesraised against the carrier protein.

In some embodiments, the carrier protein, besides its T helper, used foractivating an antibody response against the antigen of interest, couldalso activate a cytotoxic response against cells carrier peptides of thecarrier and/or stimulate an antibody response against such a carrierprotein molecule.

The carrier protein molecule could also consist in a homo-oligomer or ahomo-polymer of the native protein, from which it is derived, as well asa homo-oligomer or a homo-polymer of a peptide fragment of the nativeprotein, from which it is derived. The antigenic protein of interestcould also consist in a hetero-oligomer or a hetero-polymer comprising acombination of several distinct peptide fragments initially included inthe native protein from which it is derived.

As used herein, the expression “antigenic protein” means any protein orany peptide being at least 10 amino acid long, including a haptenpeptide, able to be specifically recognized by receptors for theantigens expressed by the B lymphocytes of a host organism, whetherhuman or animal, more particularly a mammal, such antigenic protein,once included in an immunogenic product of the invention, stimulatingthe production of antibodies recognizing said antigenic protein.

It is meant under “antigenic protein” any protein comprising one or moreB epitopes of the native antigenic protein against which the productionof antibodies if being sought. Said antigenic molecule of interest couldconsist in the native protein itself or a protein derivate of the nativeprotein, such as a peptide fragment of the native protein, as well asany biologically inactivated form of the native protein obtained throughchemical, physical treatment or genetic mutation. The antigenic moleculeof interest could also consist in a homo-oligomer or a homo-polymer ofthe native protein as well as a homo-oligomer or a homo-polymer of apeptide fragment of the native protein. The antigenic protein ofinterest could also consist in a hetero-oligomer or a hetero-polymercomprising a combination of several distinct peptide fragments initiallyincluded in the native protein.

In an immunogenic product according to the invention, advantageously,less than 30% and preferably less than 20% of antigenic proteins (i) arecovalently linked to the carrier protein molecules (ii).

In an immunogenic product according to the invention, advantageously, atleast 1%, and preferably at least 2%, of the antigenic proteins (i) arecovalently linked to the carrier molecules (ii).

It has been shown that an immunogenic product according to theinvention, such as hereinabove defined, is stable in an aqueoussolution. The stability of an immunogenic product of the invention ismore particularly characterized in that said immunogenic product has itsown isoelectric point, distinct from the isoelectric point of at leastone of its protein components, respectively the antigenic protein (i)and the carrier protein molecule (ii), and in that it therefore migratesaccording to a distinct protein strip from at least one of its proteinstrips respectively corresponding to both protein components making itup in isoelectrofocusing trials.

It has also been shown, through immunoblotting trials (“Western blot”),that the immunogenic product of the invention migrates in anelectrophorese gel, under non denaturating conditions, according to asingle protein strip, which illustrates the fact that said immunogenicproduct has the form of a homogeneous population of soluble proteinconstructions.

Moreover, it has been shown that the antigenic protein (i) as well asthe protein molecule (ii) included under the form of proteinheterocomplexes in the immunogenic product of the invention were bothrecognized by antibodies specifically recognizing each of such proteins.Thus, the immunogenic product according to the invention comprises theantigenic protein (i) and the carrier protein molecule (ii) in theirnative structure. Such a technical feature of the immunogenic productaccording to the invention is particularly advantageous for inducing animmune response against native antigens, i.e. an efficient and trulyprotective immune response of the host organism. It has been moreparticularly shown that an immunogenic product according to theinvention induces, in the host organism to which it is administered, theinduction of a strong efficient humoral response against nativeantigens, associated to the production of neutralizing or blockingantibodies, towards the deleterious biological activity of such nativeantigens.

It has been shown according to the invention, with various antigens ofinterest, that the humoral immune response obtained using an immunogenicproduct such as defined hereinabove, was 10 to 1000 times higher thanthe humoral immune response obtained with the administration of aconventional covalent conjugate between the antigen of interest and thecarrier protein molecule.

Preferably, in an immunogenic heterocomplex included in the immunogenicproduct of the invention, the plurality of antigenic proteins (i) ismade up of a plurality of specimens of a single antigenic protein.

Thus, according to a most preferred embodiment, the immunogenic productof the invention is implemented for obtaining specific antibodies raisedagainst a single antigen of interest.

It has also been shown according to the invention that an immunogenicproduct comprising immunogenic heterocomplexes such as hereinabovedefined, is particularly well adapted to the immunization of anindividual, through the production of antibodies, against a “selfantigen” of interest, i.e. against a protein being naturally produced bysaid individual, for which there exists a tolerance of the immunesystem, in particular an at least partial deletion of auxiliarylymphocyte T clones (T helper cells) specifically recognizing saidantigen.

In other words, the presentation of the “self” antigen to the cells ofsaid individual's immune system, under the form of an immunogenicproduct comprising immunogenic heterocomplexes of the invention, allowsto “break” the tolerance of the individual's immune system towards suchan antigen. Without wishing to be bound to any theory, the Applicantbelieves that the opportunity to obtain a high level of antibodyresponse against a “self” antibody is due to the presence within theheterocomplex of numerous epitopes of the “auxiliary T” type (or Thelper) carried by the carrier protein molecule, activating theauxiliary T lymphocytes, and the various cytokins produced by theactivated auxiliary T lymphocytes, including IL-2, allows to promotesome activation of the B cells to “self” antigens present in the latentstate within the organism, and to thereby break the immune tolerance ofB cells to “self” antigens.

Thus, according to a preferred embodiment, the immunogenic product ofthe invention is characterized in that the antigenic proteins (i)consists in a plurality of specimens of a protein being normallyrecognized as a self protein by the cells of said subject's immunesystem.

As the major proportion, more than 60%, of associations between theantigen of interest and the carrier protein molecule, occurs through noncovalent interactions, there exists no other theoretical limitation inthe number of molecules of the antigen of interest associated with asingle carrier protein molecule, than the steric availability of themolecules of the antigen of interest to such a carrier molecule. Inparticular, the number of molecules of the antigen of interestassociated to a single carrier protein molecule is not limited by thenumber of chemically reactive functions carried by the carrier moleculeallowing for creating covalent links with a plurality of molecules ofthe antigen of interest. Consequently, the only physical limitationseems to be the number of sites of the carrier protein molecule (ii)available to the antigenic protein (i).

For the same reasons, the size of the antigen of interest to beassociated to the carrier protein molecule is not either strictlylimited, the antigen of interest consequently being able to consist infull proteins of at least 10 kDa, such as the various cytokins, as IL-4,IL-10, VEGF as well as the alpha interferon.

Moreover, even for the antigens of interest consisting in full proteinswith a molecular mass higher than 10 kDa, an immunogenic heterocomplexof the invention can comprise an association of several antigens ofinterest on a single carrier molecule, if the size of the carriermolecule makes it possible.

When the carrier protein molecule has a small size, for example a sizelower than 10 kDa, or even lower than 5 kDa, the Applicant believes,without wishing to be bound to any theory, that the partially covalentassociations between the antigen of interest and said carrier protein,forming the protein heterocomplexes included in the immunogenic productof the invention, allow for such a conformation of heterocomplexes thatboth the antigen of interest and the carrier protein molecule areavailable to receptors of the immune system cells.

This is even an additional technical advantage provided to theimmunogenic product comprising heterocomplexes such as hereinabovedefined, as the presentation to B lymphocytes of a plurality ofspecimens on one single carrier molecule, included in the heterocomplex,enhances the “capping” phenomenon through “cross-linking” of receptorsof the B cell recognizing the antigen, contributing to the activation ofthe B cell receiving, in addition, activation signals coming fromcytokins produced by the activated auxiliary T lymphocytes activated bymeans of auxiliary T epitopes carried by carrier protein molecule.

Thus, according to a most preferred embodiment of the immunogenicproduct, the latter comprises 5 to 50 antigenic proteins (i) for onecarrier protein molecule (ii), preferably 20 to 40 antigenic proteins(i) for one carrier protein molecule (ii).

The number of molecules of the antigen of interest on one single carrierprotein molecule respectively depends on the size of the carriermolecule and on the size of the molecule of the antigen of interest. Thebigger the carrier molecule is and offers a large association surfacewith the antigen of interest, the more the immunogenic heterocomplexwill comprise, for one single of the carrier molecules it contains, ahigher number of specimens of the molecule of the antigen of interest.Similarly, the more reduced the size of the molecule of the antigen ofinterest is, the larger the number of specimens will be of the moleculeof the antigen of interest on the same carrier molecule.

By way of illustration, it has been shown according to the inventionthat when the carrier protein molecule is KLH, 20 to 40 molecules ofIL-4, IL-10, alpha interferon or VEGF are associated to each carriermolecule.

It has been shown that the solubility of the immunogenic product in anaqueous solution varies with the modification of the balances masteringthe molecular interactions within heterocomplexes, more particularly theelectrochemical balances depending on the so-called “weak” (noncovalent) links as well as the respective concentrations in antigenicproteins and the carrier protein molecule, as well as with theconditions of ionic strength, pH and temperature.

Preferably, the covalent bonds between one or more antigenic proteins(i) and the carrier protein molecule (ii) occur by means of abifunctional bonding chemical agent.

Such a chemical agent could be cyanogen bromide, glutaraldehyde,carbodiimide, or succinic anhydride.

As for carbodimides, the following compounds could be used:1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) and1-ethyl-3-(4-azonia-4,4-limethylpentyl)carbodiimide,1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)-carbodiimide,(1-ethyl-3-(3-dimethyaminopropyl carbodiimide (EDC) and1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

As homo-bifunctional coupling agents, the following compounds could beused:

-   -   N-hydroxysuccinimide, dithiobis (succinimidylpropionate) esters,        disuccinimidyl suberate, and disuccinimidyl tartrate;        bifunctional imidoesters dimethyl adipimidate, dimethyl        pimelimidate, and dimethyl suberimidate;    -   reagents with a sulphydryl,        1,4-di-[3′-(2′-pyridyledithio)propionamido]butane,        bismaleimidohexane, and bis-N-maleimido-1,8-octane;    -   bifunctional halides of the aryl type and        4,4′-difluoro-3,3′-dinitrophenylsulfone;    -   SMCC        (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate);    -   SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate);    -   SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate);    -   GMBS (N-(.gamma.-maleimidobutyryloxy)succinimide ester);    -   MPBH (4-(4-N-maleimidophenyl) hydrazide butyric acid);    -   M2C2H (4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide);    -   SMPT        (succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene);        and    -   SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

Preferably, the bonding chemical agent to be used comprises at least tworeactive aldehyde functions.

Most preferably, the bonding chemical agent is glutaraldehyde.

After the product comprising protein heterocomplexes has been formedthrough a coupling of carrier protein molecules with antigenic proteinswith the use of the bonding chemical agent, the resulting product couldbe stabilized by means of a protein stabilizing agent, such asformaldehyde, able to create intrachain bonds.

The immunogenic products comprising immunogenic heterocomplexes of theinvention have the form of microparticles soluble in solution, inparticular in an aqueous solution, their average size varying dependingon (i) the size of the carrier protein molecule, (ii) the size and thenumber of antigenic proteins associated to one single carrier proteinmolecule and (iii) the number of carrier molecules associated to theantigenic proteins present in a heterocomplex particle.

It has been found that the heterocomplex microparticles described in theexamples have an average size ranging from 100 nm to 300 nm.

Most preferably, an immunogenic product comprising immunogenic proteinheterocomplexes of the invention exclusively comprises carrier moleculesassociated to antigenic proteins, with the exclusion of any othermaterial. More particularly, a heterocomplex of the invention does notcomprise any other polymeric, proteinaceous or non proteinaceousmaterial, other than the carrier and antigenic proteins characterizingit.

Recently, ZAGURY D et al. (2001, Proc. Nail. Acad. Sci. USA.98(14):8024-8029), in a bibliographical study, suggested to induce ananti-cytokin immunity in patients in order to counteract the abnormalproduction in such pathologies of some cytokins, including interleukins,lymphokins, monokins, interferons, physiologically acting in thetissues, locally as a factor of programmed cell proliferation,differentiation, or death.

The above-mentioned authors state that the strategies of vaccine therapywere, until now, exclusively focused on the antigenic aggressor, whetherit is a micro-organism, a cell or an allergen, but never attempted tofight the deregulation of cytokins induced under the effect of theaggressor. Said authors suggest an anti-cytokin vaccination as a priorstep to a conventional vaccination having as an aim to neutralize orblock the immunotoxic effect of the stroma, and to allow for the normaloccurrence of the immune reaction adapted towards the antigenicaggressor.

Moreover, the Applicant's prior work, mentioned in the InternationalApplication published under WO 00/03732, showed that in the case of ATLleukemia, the neck of the uterus cancer and the Kaposi sarcoma,respectively, three proteins are involved in a local immunosuppressionat the level of tumors or HIV1 infected cells:

the HTLV1 virus Tax protein,

the papillomavirus E7 protein, and

the V1H-1 virus Tat protein.

The Applicant also stated that some of such immunosuppressive proteins,such as the HIV1 Tat protein and the HPV E7 protein (Strains 16 and 18)also have activating effects on vascular endothelial cells.

They therefore suggested developing anti-cancer or anti-viral vaccinescomprising a detoxicated immunogenic compound derivate of a proteincoming from cancer cells, from virus infected cells or stroma immunecells, initially immunosuppressive and/or angiogenic with a localaction, as, for example, a protein derived from the H1V1 virus Tatprotein, the HTLV1 virus Tax protein, papillomavirus E7 protein as wellas a mannan-depending lectin under an inactivated form.

Now, it has been shown according to the invention that the immunogenicproduct comprising immunogenic heterocomplexes such as hereinabovedefined allows for the induction of a strong antibody response againstthe various above-mentioned deleterious antigenic molecules.

According to a first aspect, in the immunogenic heterocomplex of theinvention, the antigenic protein(s) (i) consist(s) in cytokins naturallyproduced by said subject.

Preferably, the antigenic protein(s) (i) is/are selected frominterleukin-4, alpha interferon, gamma interferon, VEGF, interleukin-10,alpha TNF, beta TGF, interleukin-5 and interleukin-6.

According to a second aspect, the antigenic protein(s) (i) making up animmunogenic heterocomplex of the invention is/are immunosuppressive orangiogenic proteins, or proteins derived from immunosuppressive orangiogenic proteins.

Preferably, the antigenic protein(s) (i) is/are selected amongst apapillomavirus E7 protein, the VIH 1 virus Tat protein, the HTLV 1 orHTLV 2 virus Tax protein, and the self p53 protein.

According to a third aspect, the antigenic protein(s) (i) making up animmunogenic heterocomplex according to the invention is/are proteinsbeing toxic at a low dose to man or to a non human mammal. These aremore particularly various proteins being lethal to man at a dose lowerthan 1 mg, lower than 100 μg, lower than 10 μg, even lower than 1 μg.These are predominantly toxic proteins able to be used for manufacturingso-called “biological” weapons, such as ricin, botulic toxins,staphylococcus enterotoxins, as well as an anthrax toxic protein (EF,LF, PA).

The carrier protein molecule (ii) included in an immunogenic proteinheterocomplex of the invention could be a carrier moleculeconventionally used in immunology, such as KLH, ovalbumin, bovine serumalbumin (BSA), toxoid tetanos, B cholera toxin, etc.

Moreover, in an immunogenic product of the invention, the proteincarrier molecule could be selected so as to induce or stimulate, besidesthe production of T helper lymphocytes, a cytotoxic and/or humoralimmune response against itself, and its counterpart of native protein inthe host organism, respectively through the activation of cytotoxic Tlymphocytes and of B lymphocytes specific to such a carrier molecule.

Such a particular embodiment of an immunogenic product of the inventionis particularly useful when there is simultaneously sought an efficientantibody response against an immunosuppressive or angiogenic deleteriousprotein, more particularly, for producing neutralizing or blockingantibodies, and a cell immune response generated by cytotoxic Tlymphocytes raised against cells having at their surface the nativeantigen associated to Major Histocompatibility Complex (MHC) class Imolecules, for example, an antigen of a pathogen, such as the VIH1 virusor a papillomavirus, or an antigen specifically expressed in cancercells such as CEA, p53, Di12, etc.

Thus, according to this particular embodiment, the immunogenic productof the invention is characterized in that the carrier protein molecule(ii) is an immunogenic protein inducing, besides the production of Thelper lymphocytes, the production of cytotoxic T lymphocytes raisedagainst cells having at their surface said carrier protein molecule, orany peptide being derived therefrom, in association with MajorHistocompatibility Complex (MHC) class I molecules and/or the productionof antibodies by B lymphocytes raised against the carrier protein.

Thus, immunogenic products comprising immunogenic heterocomplexes of theinvention are efficient immunologic means for the active therapeuticvaccination of an individual, whether a human mammal or a non humanmammal, against a large variety of pathologies.

Illustrative examples of such immunogenic heterocomplex compositionscontained in an immunogenic product according to the invention forpreventing or treating, through an active therapeutic vaccination,various pathologies are mentioned hereinafter.

a) For Preventing or Treating AIDS:

-   -   Carrier protein molecule (ii): gp 120, gp 160, p24, p17, nef or        Tat proteins of HIV1 virus, detoxicated or stabilized if        required, immunogenic fragments of such proteins as well as an        immunogenic protein being derived therefrom (Zagury et al.,        1998).

The mimotope gp120 protein could also be used as described by Fouts etal. (2000) and by Fouts et al. (2002).

-   -   Antigenic protein (i): Tat, IFNα, IL10 and TGFβ proteins,        detoxicated if required, immunogenic fragments of such proteins,        or an immunogenic protein being derived therefrom.

b) For Preventing or Treating the Neck of Uterus Cancer:

-   -   Carrier protein molecule (ii): papillomavirus L1, L2 and E7        proteins, preferably a papillomavirus from strain 16 or 18,        detoxicated or stabilized if required, immunogenic fragments of        such proteins as well as an immunogenic protein being derived        therefrom (Le Buanec et al., 1999).    -   Antigenic protein (i): E7, IFNα, IL10, TGFβ, TNFα and VEGF        proteins, detoxicated or stabilized if required, immunogenic        fragments of such proteins as well as an immunogenic protein        being derived therefrom.

c) For Preventing or Treating ATL Leukemia Induced by the HTLV1 or 2Viruses:

-   -   Carrier protein molecule (ii): gp61 and HTLV1 or 2 virus Tax        proteins, detoxicated if required, immunogenic fragments of such        proteins as well as an immunogenic protein being derived        therefrom (Cowan et al., 1997; Mori et al., 1996).    -   Antigenic protein (i): Tax, IL10, IFNα or TGFβ, TNFα, VEGF        proteins, detoxicated if required, immunogenic fragments of such        proteins as well as an immunogenic protein being derived        therefrom.

d) For Preventing or Treating Colon Cancer:

-   -   Carrier protein molecule (ii): CEA and p53 proteins, detoxicated        if required, immunogenic fragments of such proteins as well as        an immunogenic protein being derived therefrom (Zusman et al.,        (1996)).    -   Antigenic protein (i): IFNα, TGFβ, IL10, FasL and VEGF proteins,        detoxicated if required, immunogenic fragments of such proteins        as well as an immunogenic protein being derived therefrom.

e) For Preventing or Treating Breast Cancer:

-   -   Carrier protein molecule (ii): Di12 protein, immunogenic        fragments of such a protein as well as an immunogenic protein        being derived therefrom (Yoshiji et al., 1996).    -   Antigenic protein (i): IFNα, TGFβ, IL10, FasL and VEGF proteins,        detoxicated if required, immunogenic fragments of such proteins        as well as an immunogenic protein being derived therefrom.

f) For Preventing or Treating Pancreas Cancer:

-   -   Carrier protein molecule (ii): CaSm protein, detoxicated if        required, immunogenic fragments of such proteins as well as an        immunogenic protein being derived therefrom.    -   Antigenic protein (i): VEGF and TNFα proteins, detoxicated or        stabilized if required, immunogenic fragments of such proteins        as well as an immunogenic protein being derived therefrom.

g) For Preventing or Treating Prostate Cancer:

-   -   Carrier protein molecule (i): OSA and ETS2 proteins, detoxicated        or stabilized if required, immunogenic fragments of such        proteins as well as an immunogenic protein being derived        therefrom. (Sementchenko V I et al., 1998).    -   Antigenic protein (i): IL6 and TGFb proteins, detoxicated or        stabilized if required, immunogenic fragments of such proteins        as well as an immunogenic protein being derived therefrom (Adler        et al., 1999).

h) For Preventing or Treating Some Allergies:

-   -   Carrier protein molecule (ii): it is selected amongst molecular        allogens, such as Bet v 1 (birch-tree pollen), Der p 1 (acarid)        and Fel d 1 (cat) proteins, their immunogenic peptide fragments        as well as an immunogenic protein being derived therefrom. The        Bet v 1 antigen is described more particularly by Ferreira et        al. (1993), the Der p 1 antigen is described, in particular, by        Tovey et al. (1981) and the Fel d 1 antigen is described, more        particularly by Morgensterm et al. (1991)    -   Antigenic protein (i): it induces the production of neutralizing        or blocking antibodies raised against the IL4 cytokin factor,        being mainly produced by T lymphocytes of Th2 type, orienting        the humoral immune response towards the production of IgE        isotype antibodies. According to another embodiment, the        antigenic protein (i) induces the production of neutralizing or        blocking antibodies against the IL5 cytokin factor, being mainly        produced by T lymphocytes of Th2 type.

According still another embodiment, preventing allergy could occur bymeans of an immunogenic product inducing an antibody response againstthe main basophil granulation effector, i.e. IgE isotype antibodies. Forthis purpose, the invention provides an immunogenic product comprising(i) an IgE isotype human antibody and (ii) the VIH1 inactivated Tatprotein.

i) For the Prevention Against Lethal Proteins Used in Biological Weapons

Also, an immunogenic product according to the invention could be usedfor immunizing individuals against numerous toxic products used, inparticular, in chemical and biological weapons, as for example, ricin.

Amongst the most toxic proteins against which an immunization, mainlythrough the production of antibodies, is being sought, botulic toxins,ricin, staphylococcus enterotoxins, Clostridium perfringens toxins andanthrax toxic proteins.

Generally speaking, for producing an immunogenic product according tothe invention, wherein the antigenic protein (i) is a highly toxicprotein of the above-mentioned type, a previously detoxicated protein isused under the form of a toxoid. For detoxicating the protein, beforeits use for producing an immunogenic product according to the invention,various methods could be used, and preferably one of the followingmethods consisting in:

a) treating the native toxic protein by glutaraldehyde;

b) treating the native toxic protein through the combined action offormol and glutaraldehyde; or

c) if need be, through chemical modification of His and Tyr groups bymeans of appropriate reagents, for example, through carboxymethylationof such amino acid residues.

For preventing the lethal action of toxins originating from Bacillusanthracis, as proteins, antigenic proteins (i), a detoxicated proteinoriginating from an anthrax protein selected amongst EF (“EdemaFactor”), LF (“Lethal Factor”) and PA (“Protective Antigen”) proteinsare used preferably.

For preventing the lethal actions of proteins originating fromClostridium perfringens, as the antigenic protein (i), a detoxicatedprotein originating from the Epsilon toxin of Clostridium perfringensare preferably used.

For preventing the lethal action of toxins originating form Clostridiumbotulinum, as antigenic proteins (i), a detoxicated protein originatingfrom a botulic toxin selected amongst A, B, C, D, E, F and G toxinsbeing naturally synthesized in the form a single 150 kDa polypeptidechain as well as the Hc fragment of such botulinic toxins, said fragmentHc having a molecular mass of approximately 50 kDa, are preferably used.

For producing inactivated botulic toxins, the man of the art could usetechniques known per se, more particularly those used for preparing theanterior vaccine compositions, such as those described by Fiock et al.or by Siegel et al. (Fiock, M. A., Cardella, M. A., Gearinger, N. F., J.Immunol., 1963, 90, 697-702; Siegel, L. S., J. Clin. Microbiol., 1988,26, 2351-2356).

For preventing the lethal action of toxins originating from ricin seed(Ricinus communis), as the antigenic protein (i), a detoxicated proteinoriginating from the ricin toxin, preferably the β fragment of ricin, ispreferably used.

Thus, the invention also provides an immunogenic product comprising (i)the β fragment of ricin and (ii) the KLH protein.

For purifying ricin, the man of the art could use any known technique,such as those described by Osborne et al., Kabat et al. or Kunnitz etal. (Osborne, T. B., Mendel, L B. and Harris, J. F.: Amer. J. Physiol.,1905, 14, 259-269; Kabat, E. A. Heidelberger, M. and Bezer, A. E.: J.Biol. Chem., 1947, 168, 629-; Kunnitz, M. and McDonald, M.: J. Gen.Physiol., 1948, 22, 25-Moulé, Y.: Bull. Soc. Chim. Biol., 1951, 33,1461-1467). He could also make use of the affinity chromatographypurification techniques described by Tomila et al., Nicolson et al. orOlsnes et al. (Tomila, M., Kurokawa, T., Onozaki, K. et al.,:Experientia, 1972, 28, 84-85; Nicolson, G. L. and Blaustein, J.: J.Biochim. Biophys. Acta, 1972, 266, 543-547; Olsnes, S., Salvedt, E. andPihl, A.: J. Biol. Chem., 1974, 249, 803-810). The ricin A and B chainscould be purified as described by Hedge et al. (Hedge, R. and Podder, S.K.: Eur. Biochem., 1998, 254, 596-601).

For preventing the lethal action of toxins originating fromstaphylococcus and more particularly from Staphylococcus aureus, as theantigenic protein (i), a detoxicated protein originating from a toxinselected amongst SEA (“Staphylococcal Enterotoxin A”), SEB(“Staphylococcal Enterotoxin B”), SEC (“Staphylococcal Enterotoxin C”),SED (“Staphylococcal Enterotoxin D”), SEE (“Staphylococcal EnterotoxinE”), SEG (“Staphylococcal Enterotoxin G”), SEH (“StaphylococcalEnterotoxin H”), SEI (“Staphylococcal Enterotoxin I”) and TSST-1 (“ToxicShock Syndrome Toxin-1”) is preferably used.

The above listed enterotoxins could be prepared by the man of the art bymeans of techniques described in the listed work below, relating to thedescription of each of such toxins.

SEA is synthesized in the form of a precursor enterotoxin with 257 aminoacids (Huang, I. Y., Hughes, J. L, Bergdoll, M. S. and Schantz, E. J. J.Biol. Chem. 1987, 262, 7006-7013). The mature toxin with a molecularmass equal to 27,100 Da derives from the precursor toxin through theloss of a N-terminal hydrophobic leader sequence with 24 amino acidresidues (Betley, M. J. and Mekalanos, J. J. J. Bacteriol., 1998, 170.34-41). SEA exists under 3 different isoforms through their IP.

The SEB precursor protein comprises 267 amino acids (Mr=31,400 Da) witha N-terminal signal peptide with 27 amino acids. Its binding site to thereceptor of T cells (“T-Cell Receptor” or “TCR”) encompasses the shallowcavity, whereas the class II MHC molecule is fixed on an adjacent site(Kappler, J. W., Herman, A., Clements, J. and Marrack, P.: J. Exp. Med.,1992, 175, 387-396; Papageorgiu, A. C., Trauter, H. S. and Acharya, K.R. J. Mol. Biol., 1998, 277, 61-79; Soos, J. M. and Johnson, H. M.Biochem. Biophys. Res. Commun., 1994, 201, 596-602).

SEC possesses 3 antigenically distinct sub-types: SEC 1, SEC 2 et SEC 3.The precursor proteins contains 267 amino acid residues (Houde, C. J.,Hackett, S. P. and Bohach, G. A. Mol. Gen. Genet., 1990, 220, 329-333)with a signal peptide with 27 amino acid residues (Bohach, G. A. andSchlievert, P. M.: Infect. Immun., 1989, 57, 2243-2252).

SED is made up of 258 amino acid residues with a signal peptide of 30amino acid residues. Its three-dimension structure is similar to thestructure of other bacterial superantigens.

SEE having a 26,000 Da molecular mass have 81% of AA sequence homologywith SEA.

SEG is made up of 233 amino acid residues (Munson, S. H., Tremaine, M.T., Betley, M. J. and Welch, R. A.: Infect. Immun., 1998, 66,3337-3348).

SEH has a 27,300 Da molecular mass (Su, Y. C. and Wong, A. C.: Aplil.Environ. Microbiol., 1995, 61, 1438-1443). It does not have any crossedimmunologic reaction with other enterotoxins.

SEI has a sequence comprising 218 amino acid residues. This is the toxinwith the lowest homology level with other enterotoxins

SEJ made up of 269 amino acid residues has a high AA sequence homologywith SEA, SEE and SED (64-66%).

Preferably, an immunogenic product according to the invention comprises,in combination, several antigenic proteins (i) each derived from anabove-mentioned toxic protein, for example, 2, 3, 4 or 5 antigenicproteins (i) each derived from an above listed toxic protein.

For example, an immunogenic product according to the invention forpreparing a vaccine composition intended for preventing the toxicity ofstaphylococcus enterotoxins preferably comprises 2, 3, 4 or 5 antigenicproteins (i) each derived from a staphylococcus enterotoxin.

According to a particular embodiment of an immunogenic product accordingto the invention, wherein the antigenic protein(s) (i) is/are derivedfrom highly toxic proteins for man; the carrier protein is the KLHprotein.

Thus, according to a first particular aspect of an immunogenic productof the invention, wherein the carrier protein molecule both induces theproduction of auxiliary T lymphocytes (“T helper”), of cytotoxic Tlymphocytes and of B lymphocytes specific to the carrier proteinmolecule, said carrier protein molecule (ii) is selected amongst thepapillomavirus L1, L2, and E7 proteins.

Thus, according to a second particular aspect of an immunogenic productof the invention, wherein the carrier protein molecule induces, inaddition to the production of auxiliary T lymphocytes (“T helper”), thedifferentiation of cytotoxic T lymphocytes and of B lymphocytes specificto the carrier protein molecule, said carrier protein molecule (ii) isselected amongst the HIV1 virus gp160, p24, p17, Nef and Tat proteins.

Thus, according to a third particular aspect of an immunogenicheterocomplex of the invention, wherein the carrier protein moleculeboth induces the production of auxiliary T lymphocytes (“T helper”), ofcytotoxic T lymphocytes and of B lymphocytes specific to the carrierprotein molecule, said carrier protein molecule (ii) is selected amongstCEA, p53, Di12, CaSm, OSA and ETS2 proteins.

According to a fourth particular aspect of an immunogenic product of theinvention, wherein the carrier protein molecule induces, in addition tothe differentiation of auxiliary T lymphocytes (“T helper”), theproduction of antibodies raised against the carrier protein molecule,said carrier protein molecule (ii) is selected amongst Bet v 1, Der p 1and Fel d 1 proteins.

In an immunogenic product according to the invention, the immunogenicprotein heterocomplexes are selected amongst the followingheterocomplexes, where the antigenic proteins (i), on the one hand, andthe protein carrier molecule (ii), on the other hand, are respectively:

a) (i) IL-4 and (ii) KLH;

b) (i) alpha interferon and (ii) KLH;

c) (i) VEGF and (ii) KLH;

d) (i) IL-10 and (ii) KLH;

e) (i) alpha interferon and (ii) gp 160 of VIH1;

f) (i) IL-4 and (ii) the Bet v 1 allergenic antigen; and

g) (i) VEGF and (ii) the papillomavirus E7 protein;

h) (i) the inactivated VIH1 Tat protein and (ii) the VIH1 gp120 protein;

i) i) an IgE isotype human antibody and (ii) the inactivated VIH1 Tat;

j) (i) the ricin β fragment and (ii) KLH.

Method for Preparing an Immunogenic Product Comprising ImmunogenicProtein Heterocomplexes of the Invention

Another object of the invention is also a method for preparing animmunogenic product comprising the hereinabove defined immunogenicheterocomplexes, characterized in that it comprises the following stepsof:

a) incubating the antigenic proteins (i) and the carrier molecule (ii)in a molar ratio (i):(ii) ranging from 10:1 to 50:1 in the presence of abinding chemical agent;

b) collecting the immunogenic product comprising immunogenicheterocomplexes being prepared in step a).

Preferably, the binding chemical agent is glutaraldehyde.

Most preferably, the method is further characterized in that step a) isfollowed by a stabilizing step of the product comprising the immunogenicheterocomplexes by formaldehyde, prior to step b) for recovering theheterocomplexes.

Preferably, when glutaraldehyde is used as the binding chemical agent,it is present in the coupling reaction medium in a final concentrationranging between 0.002M and 0.03M, advantageously between 0.02M and0.03M, preferably in a final concentration of 0.026M.

The coupling reaction with glutaraldehyde advantageously occurs for 20minutes to 60 minutes, preferably 30 minutes, at a temperature rangingfrom 20 to 25° C.

After the coupling step, the excess glutaraldehyde is removed, forexample, through dialysis by means of a dialysis membrane with a 3 kDacutoff threshold. The dialysis step advantageously occurs at 4° C. in abuffer adjusted to pH 7.6.

For stabilizing the product comprising the protein heterocomplexes asprepared in step a), said product could be treated in solution by theformaldehyde, for example, by formaldehyde in a final concentration of 3mM. The stabilization reaction is advantageously performed for 12 to 48hours, preferably between 20 and 30 hours, and most preferably, for 24hours. The stabilization reaction using the formaldehyde isadvantageously stopped through the addition of glycine, preferably in a0.1M concentration, for 1 hour and at a temperature ranging from 20 to25° C.

Most preferred method for preparing a stable immunogenic productcomprising immunogenic protein heterocomplexes comprising TNFa and acarrier protein.

The inventors have found that the general method disclosed above in thepresent specification, when applied to heterocomplexes comprising TNFaand a carrier protein, should be carried out according to the mostpreferred embodiments that are detailed hereunder.

Thus, a further object of the present invention consists of a method forpreparing a stable immunogenic product comprising antigenicheterocomplexes of TNFa and a carrier protein, comprising the steps of:

a) providing a liquid solution containing TNFa;

b) adding one or more antioxidant compounds to said liquid solutioncontaining TNFa of step a);

c) adding a carrier protein to the liquid solution obtained at the endof step b), so as to obtain a liquid mixture of TNFa and said carrierprotein;

d) adding glutaraldehyde to the liquid mixture obtained at the end ofstep c), so as to partially covalently conjugate TNFa molecules to saidcarrier protein and obtain heterocomplexes between TNFa and said carrierprotein;

e) removing glutaraldehyde and free molecules of both TNFa and saidcarrier protein from the solution obtained at the end of step d), so asto obtain a liquid solution containing purified heterocomplexes betweenTNFa and said carrier protein;

f) adding formaldehyde to the liquid solution obtained at the end ofstep e), and maintaining the presence of formaldehyde for a period oftime ranging from 48 hours to 240 hours;

g) adding glycine to the heterocomplexes between TNFa and said carrierprotein obtained at the end of step f), and

h) removing formaldehyde and glycine from the liquid solution obtainedat the end of step g), so as to obtain a liquid solution containingstabilized heterocomplexes between TNFa and said carrier protein; and

By performing the method described above, the resulting stableimmunogenic products are endowed with increased ability to induce theproduction of antibodies that neutralize native TNFa, when administeredto a mammal in combination with one or more immunoadjuvant compounds.

Thanks to the combined steps of the method according to the invention,the final stable immunogenic product is obtained with a highreproducibility.

Notably, it has been found that the immunogenic activity of the stableimmunogenic products prepared by performing the method according to theinvention was very reproducible, from one preparation batch to another.

As shown in the examples 37-40 herein, the stable immunogenic productsprepared by performing the most preferred method above are devoid ofdetectable TNFa biological activity, both in vitro and in vivo, sincethe LD50 dose is greater than 400 ng/ml, as expressed in TNFaconcentration.

Also, it is shown herein that beyond the induction of high anti-TNFaantibody titers, the stable immunogenic products prepared by performingthe most preferred method above induce the production of highlyneutralizing anti-TNFa antibodies having a neutralizing capacity NC50 ofmore than 1/1000.

Further, it has also been shown that the stable immunogenic productsprepared by performing the most preferred method above can be safelyadministered to mammals, since these products do not induce any adverseside effect, and particularly no inflammation nor any alteration of theorgans such as heart, lungs, liver and spleen even when administered inan amount of 4000 times the Single Human Therapeutic Dose (SHTD).

Additionally, the stable immunogenic products prepared by performing themost preferred method above induce a strong production of anti-TNFaneutralizing antibodies, specifically of the IgG isotype, in Rhesusmacaques, which shows that these stable immunogenic products are able tobreak immunological tolerance for endogenously produced proteins andinduce an effective vaccination leading to neutralization of TNFa, whichvaccination is able to prevent deleterious effects of over-production ofTNFa in all pathological situations wherein TNFa is involved.

It has been further shown that the stable immunogenic products preparedby performing the most preferred method above are able to effectivelyvaccinate mammals against TNFa-induced arthritis. Still further, it hasbeen shown that these stable immunogenic products are able to preventand/or treat TNFa-induced arthritis when used alone, with no need tocomplement the preventive or curative treatment with any additionalanti-arthritis active ingredient, like methotrexate.

Without wishing to be bound by any particular theory, the applicantbelieves that the specific combination of steps a) to h) allowspreserving the spatial conformation of the TNFa starting product whileenhancing exposure of the important antigenic epitopes of TNFa to thesolvent, so as to maximize an efficient presentation of the TNFa antigento the immunocompetent cells, when it is administered to a mammal.

Without wishing to be bound by any particular theory, the applicant alsobelieves that the specific combination of steps a) to h) allows both ahigh structural and chemical stabilization of the final immunogenicproduct. Such specific features of the resulting stable immunogenicproducts obtained by the method should allow the preparation of vaccinecompositions that are also very stable during a long time of storage.

Further, it has been found that the method of the invention allows thepreparation of a final stable immunogenic product that is endowed withno residual biological activity of the TNFa starting product. Notably ithas been found that the ED50 of the TNFa starting product was of about10 pg/mL, whereas the final stable immunogenic product obtained by themethod has an ED50 value of more than 50 ng/mL. In the same time, thefinal stable immunogenic product is endowed with a high immunogenicity.

At step a), TNFa consists of a native TNFa selected from the groupconsisting of mouse TNFa, rat TNFa, rabbit TNFa and human TNFa.Preferably, TNFa consists of human TNFa.

In certain embodiments of the method according to the invention, step b)comprises the following steps:

b1) adding one or more antioxidant compounds to said liquid solutioncontaining TNFa of step a); and

b2) adding, to the liquid solution obtained at the end of step b1); oneor more compounds that induce exposition to the solvent of thehydrophobic portions of TNFa;

It has been found according to the invention that adding one or moreantioxidant compounds, at step b) or at step b1), reduces loss ofimmunogenicity of TNFa.

Preferably, at step b), or at step b1) depending of the embodiment ofthe method, the antioxidant compounds are selected from the groupconsisting of EDTA, acetyl cysteine, ascorbic acid, ascorbyl glucoside,calcium ascorbate, sodium ascorbate, disodium ascorbyl sulfate,magnesium ascorbate, magnesium ascorbyl phosphate, caffeic acid, andcysteine.

In certain embodiments of the method, step d) comprises the followingsteps:

d1) adding glutaraldehyde to the liquid mixture obtained at the end ofstep c), so as to partially covalently conjugate TNFa molecules to saidcarrier protein and obtain heterocomplexes between TNFa and said carrierprotein; and

d2) adding one or more antioxidant compounds to the heterocomplexesbetween TNFa and said carrier protein obtained at the end of step d1);

The antioxidant compounds used at step d2) are preferably the same asthose used in step b), or step b1).

At step h), formaldehyde and glycine are removed from the solutionobtained at the end of step g), so as to obtain a purified stableimmunogenic product comprising heterocomplexes between TNFa and saidcarrier protein.

At the end of step h), the stable immunogenic product is obtained underthe form of a translucent colorless liquid solution that is practicallyfree of any visible particles.

In certain embodiments, said method may also comprise a further step i)of freezing the solution obtained at the end of step h).

In certain other embodiments, said method may also comprise a furtherstep i) of lyophilizating the solution obtained at the end of step h),so as to obtain a white powder that can be stored for a long period oftime before use.

In preferred embodiments of step a), TNFa concentration ranges from 0.1mg/mL to 50 mg/mL, and even more preferably ranges from 0.5 mg/mL to 10mg/mL.

In preferred embodiments of step b), when the antioxidant compoundconsists of EDTA, final EDTA concentration ranges from 1 mM to 10 mM.

Advantageously, EDTA is added as a buffer solution having a pH rangingfrom 7 to 8.5, more preferably from 7.5 to 8.1.

At step b2) the one or more compounds that induce exposition to thesolvent of the hydrophobic portions of TNFa is most preferably DMSO.

In preferred embodiments of step b2), when the compound that inducesexposition to the solvent of the hydrophobic portions of TNFa consistsof DMSO, then final DMSO concentration ranges from 5% v/v to 20% v/v.

Advantageously, step b2) is carried out for a period of time rangingfrom 10 min to 50 min, more preferably from 20 min to 40 min.

In preferred embodiments of step c), the molar ratio of TNFa to saidcarrier protein ranges from 5:1 to 100:1, and even more preferablyranges from 20:1 to 80:1. In the most preferred embodiments of step c),the molar ratio of TNFa to said carrier protein ranges from 30:1 to70:1.

In preferred embodiments of step d), final glutaraldehyde concentrationranges from 0.05% w/w to 0.5% w/w.

Advantageously, step d2) is carried out for a period of time rangingfrom 30 min to 60 min, more preferably from 40 min to 50 min.

In preferred embodiments of step d2), when the antioxidant compoundconsists of EDTA, then final EDTA concentration ranges from 1 mM to 10mM.

In preferred embodiments of step e), glutaraldehyde is removed byperforming a dialysis or by performing an ultrafiltration withdiafiltration, or by performing Tangential Flow Filtration (TFF), asshown in the examples herein.

When dialysis is performed, a dialysis membrane with a cut-off thresholdof 6 to 8 KDa is preferably used.

In certain embodiments, the dialysis step comprises one or moresub-steps, typically three sub-steps. Preferably, dialysis comprises twoidentical sub-steps performed with the working buffer (100 mM phosphate,150 mM NaCl at pH 7.8, EDTA mM), which are followed by the thirdsub-step performed with PBS buffer.

Usually, the dialysis step is performed with a conventional buffersolution, such as a Phosphate Buffer Saline solution (PBS), against 200to 400 times the volume of the liquid solution containing theheterocomplexes between TNFa and the carrier protein, wherein the TNFaare partly covalently conjugated to the carrier protein which is used.

When ultrafiltration with diafiltration is performed, a filteringmembrane with a cut-off threshold of 10 000 Kda is preferably used, itbeing understood that the heterocomplexes between TNFa and the carrierprotein are found in the ultrafiltration retentate. Usually, the liquidused for diafiltration consists of a buffer solution, such as aPhosphate Buffer Saline solution (PBS). Usually, the diafiltration isperformed three times with an equal volume of said buffer solution.

In preferred embodiments of step f), the final concentration offormaldehyde ranges from 1% w/w to 10% w/w, and even more preferablyranges from 2% w/w to 5% w/w.

In further preferred embodiments of step f), the presence offormaldehyde is maintained during a period of time ranging from 96 hoursto 192 hours.

It has been found according to the invention that maintaining thepresence of formaldehyde with the heterocomplexes for a time period ofless than 96 hours led to a final immunogenic compound that wassignificantly less stable with time, when compared with a stableimmunogenic compound obtained according to the preferred embodiments ofthe method according to the invention.

On the other hand, it has been found according to the invention thatmaintaining the presence of formaldehyde with the heterocomplexes for atime period of more than 192 hours led to a final immunogenic compoundthat was highly stable but with a significantly lowered ability toinduce antibodies having a high neutralizing activity against nativeTNFa.

More preferably, at step f), the presence of formaldehyde is maintainedduring a period of time ranging from 120 hours to 168 hours.

Most preferably, at step f), the presence of formaldehyde is maintainedduring a period of time ranging from 130 hours to 150 hours.

In preferred embodiments of step g), formaldehyde is removed byperforming a dialysis or by performing an ultrafiltration withdiafiltration.

Advantageously, step f) is carried out at a temperature ranging from 30°C. to 42° C., more preferably from 35° C. to 39° C.

The conditions for dialysis or ultrafiltration with diafiltration thatare used in step h) are usually the same as those previously defined forstep e) above.

In preferred embodiments of step g), final glycine concentration rangesfrom 0.01 M to 10 M, and even more preferably ranges from 0.05 M to 2 M.

Advantageously, when carrying out step g) the pH of the liquid solutionis adjusted to a pH ranging from 6.8 to 7.8, more preferably from 7.0 to7.6, for example by using a base such as NaOH.

In preferred embodiments of step h), formaldehyde and glycine areremoved by performing a dialysis or by performing an ultrafiltrationwith diafiltration.

The conditions for dialysis or ultrafiltration with diafiltration thatare used in step h) are usually the same as those previously defined forstep e) above.

At step h), formaldehyde and glycine may be removed by performing a stepof dialysis, a step of ultrafiltration with dispersion or a step oftangential Flow Filtration (TFF), as shown in the examples herein.

According to the method of the invention, a wide diversity of carrierproteins known to the one skilled in the art may be used at step c). Thecarrier protein should bear sufficient helper T-cell epitopes so as toactivate T-helper and B cells and induce these cells to release enoughIL-1 and IL-2 to induce the expansion of the B cell clones that willproduce the neutralizing anti-TNFa antibodies.

A “carrier protein molecule”, included in the stable immunogenic productof the invention, means any protein or peptide being at least 15 aminoacids long, whatever its amino acid sequence, and which, when partiallycovalently being associated to the molecules of TNF a for formingprotein heterocomplexes making up the immunogenic product of theinvention, allows for a large number of molecules of TNFa to bepresented to the B lymphocytes.

According to a first aspect, the carrier protein molecule consists ofone protein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising one or more auxiliary T epitopes(“helper”) able to activate auxiliary T lymphocytes (“T helper”) of thehost organism for producing cytokines, including interleukin 2, suchcytokins, in turn, activating and inducing the proliferation of Blymphocytes, which, after maturation, will produce antibodies raisedagainst the antigenic protein (i).

According to a second aspect, a carrier protein molecule consists of oneprotein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”), as described in the above-mentioned first aspect,one or more cytotoxic T epitopes, able to induce a cell immune responsethrough the production of cytotoxic T lymphocytes specific of thecarrier protein molecule, such lymphocytes being able to specificallyrecognize cells expressing on their surface said carrier protein or anypeptide being derived therefrom, in association with class 1Histocompatibility Major Complex (HMC) molecules. If need be, thecarrier protein molecule consists in one oligomer of one protein or onepeptide, further comprising besides one or more T helper epitopes, oneor more above defined cytotoxic T epitopes.

According to a third aspect, a carrier protein molecule consists in oneprotein or one peptide being at least 15 amino acid long, as well as oneoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”) as defined in the first aspect, one or more Bepitopes, able to induce the production of antibodies by lymphocytesraised against the carrier protein.

In some embodiments, the carrier protein, besides its T helper, used foractivating an antibody response against the antigen of interest, couldalso activate a cytotoxic response against cells carrier peptides of thecarrier and/or stimulate an antibody response against such a carrierprotein molecule.

The carrier protein molecule could also consist in a homo-oligomer or ahomo-polymer of the native protein, from which it is derived, as well asa homo-oligomer or a homo-polymer of a peptide fragment of the nativeprotein, from which it is derived. The antigenic protein of interestcould also consist in a hetero-oligomer or a hetero-polymer comprising acombination of several distinct peptide fragments initially included inthe native protein from which it is derived.

Examples of protein carriers which may be used when performing themethod according to the invention include the Diphtheria and Tetanustoxoids (DT, DT CRM197, other DT mutants, e.g. position Glu-148, etc.[see, e.g., U.S. Pat. No. 4,709,017, WO93/25210, WO95/33481, etc.] andTT (and TT fragment C) respectively), Keyhole Limpet Haemocyanin (KLH),OMPC from N. meningitidis, and the purified protein derivative ofTuberculin (PPD).

The function of the carrier is to provide cytokine help in order toenhance the immune response against TNFa. A non-exhaustive list ofcarriers which may be used in the present invention include: Keyholelimpet Haemocyanin (KLH), serum albumins such as bovine serum albumin(BSA), inactivated bacterial toxins such as tetanus or diphtheria toxins(TT and DT), or recombinant fragments thereof (for example, Domain 1 ofFragment C of TT, or the translocation domain of DT), or the purifiedprotein derivative of tuberculin (PPD).

In an embodiment of the method, the carrier is Protein D fromHaemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD-bindingprotein from Haemophilus influenzae and has been patented by Forsgren(WO 91/18926, granted EP 0 594 610 B1). In some circumstances, forexample in recombinant immunogen expression systems it may be desirableto use fragments of protein D, for example Protein D ⅓.sup.rd(comprising the N-terminal 100-110 amino acids of protein D (WO99/10375; WO 00/50077)).

Thus, in preferred embodiments of the method, said carrier protein isselected form the group consisting of diphtheria toxoid (DT) and mutantsthereof, Tetanus toxoid (TT), Keyhole Limpet Haemocyanin (KLH), and thepurified protein derivative of Tuberculin (PPD), OMPC from N.meningitidis, the purified protein derivative of Tuberculin (PPD),bovine serumalbumin (BSA) and Protein D from Haemophilus influenzae.

Most preferably, said carrier protein consists of Keyhole LimpetHaemocyanin (KLH).

The present invention also relates to a method for preparing a vaccinecomposition comprising the steps of:

a) preparing a stable immunogenic product comprising antigenicheterocomplexes of TNFa by performing the method of any one of claims 1to 22; and

b) combining said stable immunogenic product comprising antigenicheterocomplexes of TNFa prepared at step a) with one or moreimmunoadjuvants.

This invention also pertains to a stable immunogenic product comprisingantigenic heterocomplexes of TNFa and a carrier protein which has one ormore of the following technical features:

i) it possesses a molecular weight ranging from 50 kDa to 8 Mda, withabout 30% of the total protein amount having a molecular weight lowerthan 1 MDa;

ii) it exhibits a molar ratio of TNFa to said carrier protein rangingfrom 40:1 to 60:1;

iii) it induces the production of anti-TNFa antibodies having a TNFaneutralizing capacity (NC50) of more than 1/1000.

By way of illustration, it has been shown according to the invention,using the first or the second above described quantification methodsthat: in the immunogenic product comprising heterocomplexes between theKLH carrier molecule and human TNFa molecules, less than 40% of the TNFamolecules are covalently linked to the KLH carrier protein molecule.

The Compositions Comprising an Immunogenic Product ComprisingImmunogenic Protein Heterocomplexes of the Invention

Another object of the present invention is also to provide a compositioncomprising an immunogenic product such as hereinabove defined.

The invention also relates to a pharmaceutical composition comprising aprotein immunogenic product such as hereinabove defined.

Another object of the invention is an immunogenic compositioncharacterized in that it comprises, as the active ingredient, animmunogenic product as hereinabove defined, in association with one ormore physiologically compatible excipients.

It also relates to a vaccine composition characterized in that itcomprises, as the active ingredient, an immunogenic product ashereinabove defined, in association with one or more physiologicallycompatible excipients.

Depending on the target objectives, systemic adjuvants or mucosaladjuvants are being used. For example, a mucosal adjuvant is preferablyused for preventing the epithelial tissue cancers and preferablysystemic adjuvants are used for preventing or treating virus infectionssuch as HIV1 and HTLV1 as well as for preventing or treating allergies.

Amongst systemic adjuvants, those of the IFA type are preferably used(Incomplete Freund's Adjuvant), as well as calcium phosphate or aluminahydroxide.

Amongst mucosal adjuvants, those preferably used are like B chloratoxin(CTB) or a mutant of the LT toxin (LTμ).

According to a particular aspect, an immunogenic composition accordingto the invention also comprises one or more immuno-stimulating agents,in combination with an immunity adjuvant, such as for example, the CpGimmuno-stimulating agent well known in the state of the art.

It has indeed been shown according to the invention that the use of theCpG adjuvant, and more particularly the CpG adjuvant wherein theintra-chain bonds between nucleotides consist in phosphorothioate bondsto stimulate the simultaneous production of IgG and IgA isotypeantibodies, after a systemic administration.

It also relates to a mucosal or systemic vaccine, characterized in thatit comprises, as the active ingredient, an immunogenic product such ashereinabove defined, in association with one or more excipients,including physiologically compatible adjuvants.

The immunogenic compositions or the vaccines according to the presentinvention are useful for example in the treatment, both curative andpreventive, of cancers, more particularly, of cancers induced by virusessuch, as for example, the ATL (Acute T cell leukemia) caused by theHTLV1, or the neck of uterus cancer caused by the papillomavirus, aswell as the Burkitt lymphoma as well as the Kaposi sarcoma caused by theviruses from the herpes family, respectively the Epstein-Barr (EBV) andthe HHV8 as well as in treatment of AIDS or for preventing or treatingallergic reactions.

The immunogenic products according to the invention could be used asfollows.

To a patient, is administered, under a form adapted to the systemic ormucosal administration, an immunogenic product comprising immunogenicprotein heterocomplexes according to the present invention, for example,intranasally, in a sufficient amount to be therapeutically efficient, toa subject in need of such a treatment. The dose to be administered couldrange for example from 10 to 1000 μg intranasally, once a week for 2months and then, given the transitory character of the antibody responsedirected against the antigen of interest, periodically depending on theserum antibody rate, for example, once every 2 to 6 months.

Two or more different immunogenic products could be administered in onesingle preparation for inducing neutralizing antibodies in all thedeleterious functional sites should one single molecule not carry allthe active sites of the overproduced toxin or cytokin which is to beneutralized.

As for drugs, the immunogenic products of the invention could beincorporated into pharmaceutical compositions adapted for anadministration through the systemic route or an administration throughthe mucosal route, including the oromucosal route, more particularly,the intranasal route, the oral route and the vaginal route. Theadministration could be performed in one single dose or a dose repeatedonce or several times after some time interval.

This is why the present application has also as an object apharmaceutical, curative, or preventive composition, characterized inthat it comprises as an active ingredient, one or more immunogenicproducts such as hereinabove defined. The immunogenic product could bepackaged alone or mixed with an excipient or a mixture ofpharmaceutically acceptable excipients such as an adjuvant. Amongst theexcipients adapted for the intranasal or oral route, are particularly tobe selected the capryl caproyl macrogol glycerides as Labrasol® from theGATTEFOSSE corporation or alumina hydroxide (Alhydragel, Superfos,Denmark).

For the oral administration according to the invention, the activeingredient will be associated to a mucosal immunity adjuvant, such as aCT, LT or CTB mutant.

Galenic forms are particularly well suited, as described by Boyaka etal. “Strategies for mucosal vaccine development” in Am. J. Trop. Med.Hyg. 60(4), 1999, pages 35-45. Are also to be mentioned gastroresistant, more particularly bioadhesive microgranules, such asdescribed by Rojas et al. in Pharmaceutical Research, vol. 16, no 2,1999, page 255.

Under the particular implementing conditions, an above-mentioned vaccinepharmaceutical composition will be selected, characterized in that itcomprises a mucosal immunity adjuvant, such a CT mutant (cholera toxin)or a LT mutant E. coli labile enterotoxin).

Under other particular implementing conditions, a vaccine pharmaceuticalcomposition will be selected, characterized in that it contains anadjuvant absorbing the active ingredient, such as alumina hydroxide orgold particles.

Another object of the present invention is a method for preparing acomposition as described hereinabove, characterized in that are mixed,using methods known per se, the active immunogenic product(s) with theacceptable excipients, including pharmaceutical acceptable ones and ifneed be, with a systemic or mucosal immunity adjuvant.

Under preferred implementing conditions of the above-mentioned method,bioadhesive and gastroresistant microgranules are prepared for thedigestive route containing the immunogenic active ingredients and, ifneed be, the adjuvants.

Most Preferred Embodiments of a Vaccine Composition According to theInvention.

To prepare the vaccine composition above, the stable immunogenic productobtained by the method according to the invention is adjusted to anappropriate concentration, optionally combined with a suitable vaccineadjuvant, and packaged for use.

As used herein, the term “adjuvant” refers to its ordinary meaning ofany substance that enhances the immune response to an antigen with whichit is mixed. Adjuvants useful in the present invention include, but arenot limited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvumare potentially useful adjuvants

Any adjuvant known in the art may be used in the vaccine compositionabove, including oil-based adjuvants such as Freund's Complete Adjuvantand Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g.,trehalose dimycolate), bacterial lipopolysaccharide (LPS),peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such asN-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g.,extracted from Klebsiella pneumoniae), streptococcal preparations (e.g.,OK432), Biostim™ (e.g., 01K2), the “Iscoms” of EP 109 942, EP 180 564and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils(such as miglyol), vegetable oils (such as arachis oil), liposomes,Pluronic.RTM. polyols, the Ribi adjuvant system (see, for example GB-A-2189 141), or interleukins, particularly those that stimulate cellmediated immunity. An alternative adjuvant consisting of extracts ofAmycolata, a bacterial genus in the order Actinomycetales, has beendescribed in U.S. Pat. No. 4,877,612. Additionally, proprietary adjuvantmixtures are commercially available. The adjuvant used will depend, inpart, on the recipient organism. The amount of adjuvant to administerwill depend on the type and size of animal. Optimal dosages may bereadily determined by routine methods.

Suitable adjuvants include but are not limited to surfactants, e.g.,hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′-N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polyanions, e.g.,pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides,e.g., muramyl dipeptide, MPL, aimethylglycine, tuftsin, oil emulsions,alum, and mixtures thereof. Other potential adjuvants include the Bpeptide subunits of E. coli heat labile toxin or of the cholera toxin.McGhee, J. R., et al., “On vaccine development,” Sem. Hematol., 30:3-15(1993).

The adjuvant properties of saponin have been long known, as has itsability to increase antibody titres to immunogens. As used herein, theterm “saponin” refers to a group of surface-active glycosides of plantorigin composed of a hydrophilic region (usually several sugar chains)in association with a hydrophobic region of either steroid ortriterpenoid structure. Although saponin is available from a number ofdiverse sources, saponins with useful adjuvant activity have beenderived from the South American tree Quillaja saponaria (Molina).Saponin from this source was used to isolate a “homogeneous” fractiondenoted “Quil A” (Dalsgaard, K., (1974), Arch. Gesamte Virusforsch.44:243).

Dose-site reactivity is a major concern for both the veterinary andhuman use of Quil A in vaccine preparations. One way to avoid thistoxicity of Quil A is the use of an immunostimulating complex (known asan ISCOM™, an abbreviation for Immuno Stimulating COMplex). This isprimarily because Quil A is less reactive when incorporated intoimmunostimulating complexes, because its association with cholesterol inthe complex reduces its ability to bind to cholesterol in cell membranesand hence its cell lytic effects. In addition, a lesser amount of Quil Ais required to generate a similar level of adjuvant effect.

The immunomodulatory properties of the Quil A saponins and theadditional benefits to be derived from these saponins when they areincorporated into an immunostimulating complex have been described invarious publications, e.g. Cox and Cox, J. C. and Coulter, A. R.Advances in Adjuvant Technology and Application in Animal ParasiteControl Utilising Biotechnology, Chapter 4, Editor Yong, W. K. CRC Press(1992); Cox, J. C. and Coulter, A. R. (1997) Vaccine, 15(3):248-256;Cox, J. C. and Coulter, A. R. (1999) BioDrugs 12(6):439-453); Dalsgaard,(1974) (supra); Morein et al., (1989) “Immunostimulating complex(ISCOM)”, In “Vaccines: Recent Trends and Progress”. G. Gregoriadis, A.C. Allison and G. Poster (Eds). Plenum Press, New York, p. 153;Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067.

Classic ISCOMs are formed by combination of cholesterol, saponin,phospholipid, and immunogens, such as viral envelope proteins. ISCOMmatrix compositions (known as ISCOMATRIX™) are formed identically, butwithout viral proteins. ISCOMs appear to stimulate both humoral andcellular immune responses. ISCOMs have been made with proteins fromvarious viruses, including HSV-1, CMV, EBV, hepatitis B virus (HBV),rabies virus, and influenza virus see for example, I. G. Barr et al.,Adv. Drug Delivery Reviews, 32:247-271 (1998). It has been observed thatwhere naked DNA or polypeptides from infectious agents are poorlyimmunogenic when given by themselves, inclusion within ISCOMs hasincreased their immunogenicity. Various proteins formulated with ISCOMshave been shown to induce CTL, mainly in rodent models. Berzofsky,(1991), Biotechnol. Ther. 2:123-135; Hsu et al., (1996), Vaccine14:1159-1166; Lipford et al., (1994), Vaccine 12:73-80; Mowat et al.,(1991), Immunology 72:317-322; Osterhaus et al., (1998), Dev. Biol.Stand. 92:49-58; Rimmelzwaan et al., (1997), J. Gen. Virol. 78 (pt.4):757-765; Sambhara et al., (1998), J. Infect. Dis. 177:1266-1274;Sambhara et al., (1997), Mech. Aging Dev. 96:157-169; Sjolander et al.,(1997), Vaccine 15:1030-1038; Sjolander et al., (1998), J. Leukoc. Biol.64:713-723; Takahashi et al., (1990), Nature 344:873-875; Tarpey et al.,(1996), Vaccine 14:230-236; Trudel et al., (1987), Vaccine 10: 107-112;Verschoor et al., (1999), J. Virol. 73:3292-3300; Villacres-Eriksson,(1995), Clin. Exp. Immunol. 102:46-52; Zugel et al., (1995), Eur. J.Immunoll. 25:451-458.

Association between a stable immunogenic product obtained by the methodof the invention and an adjuvant is thought to be important for optimalinduction of immune responses. A number of studies have been done whichconfirm this hypothesis including work with virosomes and ISCOMs™(Ennis, F. A., Crux, J., Jameson, J., Klein, M., Burt, D. andThipphawong, J. 1999. Virology 259: 256-261, Zurbriggen, R.,Novak-Hofer, I., Seelig, A. and Gluck, R. (2000), Progress in lipidResearch 39: 3-18, Voeten, J. T. M., Nieuwkoop, N. J.,Lovgren-Bengtsson, K., Osterhaus, D. M. E. and Rimmelzwaan, G. F. 2000.Euro J 1 mm (Submitted)). Typically association between ISCOM™ andantigen has been achieved by incorporation of amphipathic antigens intothe ISCOM™ structure during formation (Morein, B., B. Sundquist, S.Hoglund, K. Dalsgaard, and A. Osterhaus. 1984. Nature 308:457).Incorporation was by hydrophobic interactions. More recently methods toassociate antigens with a preformed protein-free immunostimulatingcomplex (ISCOMATRIX™) utilizing chelating and electrostatic interactionshave been developed (International Patent Applications Nos.PCT/AU98/00080-WO 98/36772, and PCT/AU00/00110).

In certain embodiments of a vaccine composition according to theinvention, said vaccine composition comprises, as pharmaceuticalexcipients, one or more charged inorganic carriers. Examples of chargedorganic carriers which are adjuvants suitable for use in the presentinvention include, but are not limited to, saponin, saponin complexes,any one or more components of the immunostimulating complex of saponin,cholesterol and lipid known as ISCOMATRIX™ (for example the saponincomponent and/or the phospholipid component), liposomes or oil-in-wateremulsions. (The composition and preparation of ISCOMATRIX™ is describedin detail in International Patent Application Number PCT/SE86/00480,Australian Patent Numbers 558258 and 632067 and European PatentPublication No. 0 180 564, the disclosures of which are incorporatedherein by reference). Further examples of adjuvants include, but are notlimited to, those detailed in the publications of Cox and Coulter, 1992,1997 and 1999. It should be understood that the subject organic carriermay be naturally occurring or it may be synthetically or recombinantlyderived.

The vaccine compositions may also include further adjuvants to enhanceeffectiveness of the composition. Suitable adjuvants include, but arenot limited to: (1) aluminum salts (alum), such as aluminum hydroxide,aluminum phosphate, aluminum. sulfate, etc; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (PCT Publ. No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles, (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%pluronic-blocked polymer, and thr-MDP (see below) either microfluidizedinto a submicron emulsion or vortexed to generate a large particle sizeemulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, suchas Stimulon™ (Cambridge Bioscience, Worcester, Mass.); (4) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc), interferons (e.g. gamma interferon), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, e.g.WO 93/13302 and WO 92/19265; (7) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition; and (8) microparticles with adsorbed macromolecules, asdescribed in International Patent Application No. PCT/US99/17308. Alumand MF59 are preferred.

As mentioned above, suitable muramyl peptides include, but are notlimited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normauramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Further adjuvants for inducing a mucosal or a systemic response againstan immunogenic compound according to the invention may be those that aredescribed in the book of Vogel et al. (Vogel F. R., Powell M. F. andAlving C. R., “A compendium of vaccine adjuvants and excipients”; 2^(nd)Edition; Vogel F. R. and Powell M F, 1995, “A summary compendium ofvaccine adjuvants and excipients. In: Powell M F, Newman M J eds.“Vaccine design: the subunit and adjuvant approach”. New York: Plenumpublishing, 1995: 141-228).)

Generally, adjuvants that may be comprised in a vaccine compositionaccording to the invention encompass, without being limited to:

(i) Gel-type adjuvants, such as aluminum hydroxide or aluminum phosphate(Glenny A T et al., 1926; J Pathol Bacteriol; Vol. 29: 38-45; Gupta R Kand Siber G R, 1994; Biologicals; Vol. 22: 53-63);

(ii) Microbial adjuvants, such as:

-   -   DNA CpG motifs (Chu R S et al., 199; J Exp Med; Vol. 186:        1623-1631); Monophosphoryl lipid A (Schneerson R et al., 1991; J        Immunol; Vol. 147: 2136-2140); Cholera toxin (Holmgren J et al.,        1993; Vaccine; Vol. 11: 1179-1184; Okahashi N et al., 1996;        Infect Immun; Vol. 64: 1516-1525);    -   E. coli heat-labile toxin (Lycke N et al., 1992; Eur J Immunol;        Vol. 22: 2277-2281; de Haan L et al., 1996; Vaccine; Vol. 14:        260-266; Chong C et al., 1998. Vaccine; Vol. 16: 732-740);    -   Pertussis toxin (Roberts M et al., 1995; Infect Immun; Vol. 63:        2100-2108; Mu H H and Sewell W A, 1994; Immunology; Vol. 83:        639-645);    -   Muramyl dipeptide (Ellouz F et al.; 1974; Biochem Biophys Res        Commun; Vol. 59: 1317-1325); Cohen L Y et al., 1996; Cell        Immunol; Vol. 169: 75-84);

(iii) Oil-emulsion and emulsifier-based adjuvants, such as:

-   -   Freund's incomplete adjuvant (Dhiman N et al., 1997; Med        Microbiol Immunol (Berlin); Vol. 186: 45-51; Putkonen P et al.,        1994; J Med Primatol; Vol. 23: 89-94);    -   MF59 (Dupuis M et al., 1998; Cell Immunol; Vol. 186: 18-27; Kahn        J O et al., 1994; J infect Dis; Vol. 170: 1288-1291; Ott G et        al., 1995; Vaccine. Vol. 13: 1557-1562);    -   SAF (Allison A C; 1998; Dev Biol Stand; Vol. 92: 3-11; Gupta R K        et al., 1993; Vaccine; Vol. 11: 293-306; Byars N E et al., 1994;        Vaccine; Vol. 12: 200-209);

(iv) Particulate adjuvants, such as:

-   -   Immunostimulatory Complexes (ISCOMs) (Putkonen P et al., 1994; J        Med Primatol; Vol. 23: 89-94; Gupta R K et al., 1993; Vaccine;        Vol. 11: 293-306; Sjolander A et al., 1997; Cell Immunol; Vol.        177: 69-76);    -   liposomes (Richards R L et al., 1998; Infect Immun; Vol. 66:        2859-2865; Fernandes I et al., 1997; Mol Immunol; Vol. 34:        569-576);    -   biodegradable microspheres (Men Y et al., 1996; Vaccine; Vol.        14: 1442-1450; Shahin R et al., 1995; Infect Immun; Vol. 63:        1195-1200;    -   saponins (QS-21) (Newman M J et al., 1992; J Immunol; Vol. 148:        2357-2362; Neuzil K M et al., 1997; Vaccine. Vol. 15: 525-532);

(v) Synthetic adjuvants, such as:

-   -   nonionic block copolymers (Hunter R L et al., 1994; AIDS Res Hum        Retroviruses; Vol. 10 (Suppl 2); S95-S98; Newman M J et al.,        1997; Mech Ageing dev; Vol. 93: 189-203);    -   muramyl peptide analogues (Cohen L Y et al., 1996; Cell Immunol;        Vol. 169: 75-84; Fast D J and Vosika G J, 1997; Vaccine; Vol.        15: 1748-1752; Bahr G M et al., 1995; Int J Immunopharmacol;        Vol. 17: 117-131);    -   polyphosphazene (Payne L G et al., 1998; Vaccine; Vol. 16:        92-98);    -   synthetic polynucleotides (Johnson A G, 1994; Clin Microbiol        Rev; Vol. 7: 277-289; Harrington D G et al., 1979; Infect Immun;        Vol. 24: 160-166); and

(vi) cytokines, such as:

-   -   IFN-g (Odean M J et al., 1990; Infect Immun; Vol. 58: 427-432);    -   Interleukin-2 (Nunberg J et al., 1989; Proc Natl Acad Sci USA;        Vol. 86: 4240-4243);    -   Interleukin-12 (Luis C et al., 1994; Science; Vol. 263: 235-237;        Bliss J et al., 1996; J Immunol; Vol. 156: 887-894; Jankovic D        et al., 1997, J Immunol; Vol. 159: 2409-2417).

A further aspect of the present invention therefore relates to the useof a stable immunogenic product or of a vaccine composition as definedabove to induce an immune response in a mammal including a humoralimmune response wherein antibodies that neutralize theimmunosuppressive, apoptotic or angiogenic properties of the nativecytokine.

A further object of the invention consists of a method for inducing theproduction of antibodies that neutralize the activity of native TNFa ina mammal, comprising a step of administering to said mammal (i) avaccine composition according to claim 23 or (ii) a stable immunogenicproduct comprising antigenic heterocomplexes of TNFa and a carrierprotein according to claim 24 together with one or more immunoadjuvants.

The vaccine compositions optionally may include vaccine-compatiblepharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluents that serve as pharmaceutical vehicles,excipients, or media. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma.

A vaccine composition according to the invention may also further one ormore pharmaceutically acceptable carriers and/or diluents, such carriersinclude any carrier that does not itself induce the production of aresponse harmful to the individual receiving the composition. Suitablecarriers are typically large, slowly metabolized macromolecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates (such asoil droplets or liposomes), and inactive virus particles. Such carriersare well known to those of ordinary skill in the art.

The vaccine compositions according to the invention may typically alsocontain diluents, such as water, saline, glycerol, ethanol, etc.Additionally, auxiliary substances, such as wefting or emulsifyingagents, pH buffering substances, and the like, may be present in thecompositions.

Suitable preparations of the vaccines of the present invention includeinjectables, either liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, a liquid pharmaceuticallyacceptable carrier prior to injection may also be prepared. The vaccinepreparation may be emulsified. Additional substances that can beincluded in a product for use in the present methods include, but arenot limited to one or more preservatives such as disodium or tetrasodiumsalt of ethylenediaminetetracetic acid (EDTA), merthiolate, and thelike.

The vaccine compositions optionally may include vaccine-compatiblepharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluents that serve as pharmaceutical vehicles,excipients, or media. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma.

The vaccine compositions can be packaged in forms convenient fordelivery. The compositions can be enclosed within a capsule, caplet,sachet, cachet, gelatin, paper, or other container. These delivery formsare preferred when compatible with entry of the immunogenic compositioninto the recipient organism and, particularly, when the immunogeniccomposition is being delivered in unit dose form. The dosage units canbe packaged, e.g., in tablets, capsules, suppositories or cachets.

The forms suitable for injectable use include sterile aqueous solutions(where water soluble) or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. They must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active immunogenic compound according to the invention issuitably protected, it may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, compressed into tablets,or incorporated directly with the food of the diet. For oral therapeuticadministration, the active immunogenic compound according to theinvention may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such vaccine compositions andpreparations should contain at least 1% by weight of active immunogeniccompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 5 to about 80%of the weight of the unit. The amount of active immunogenic compound insuch therapeutically useful vaccine compositions is such that a suitabledosage will be obtained. Preferred vaccine compositions or preparationsaccording to the present invention are prepared so that a dosage unitform contains between about 0.1 g and 2000 mg of active immunogeniccompound.

A vaccine composition according to the invention suitable for oraladministration may also be prepared under the form of a liquid solution,including a liquid aerosol formulation.

The liquid aerosol formulations contain the immunogenic product and adispersing agent in a physiologically acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of the immunogenic product and a dispersing agent.With either the liquid or dry powder aerosol formulation, theformulation must be aerosolized. That is, it must be broken down intoliquid or solid particles in order to ensure that the aerosolized doseactually reaches the mucous membranes of the nasal passages or the lung.The term “aerosol particle” is used herein to describe the liquid orsolid particle suitable for nasal or pulmonary administration, i.e.,that will reach the mucous membranes. Other considerations, such asconstruction of the delivery device, additional components in theformulation, and particle characteristics are important. These aspectsof pulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art. In a particular embodiment, the mass mediandynamic diameter will be 5 micrometers or less in order to ensure thatthe drug particles reach the lung alveoli [Wearley, L. L., Crit. Rev. inTher. Drug Carrier Systems 8:333 (1991)].

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,(Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22)and can be used in connection with the present invention.

The vaccine compositions according to the invention may be administeredto the subject to be immunized by any conventional method including,e.g., by oral, intravenous, intradermal, intramuscular, intramammary,intraperitoneal, or subcutaneous injection; by oral, transdermal,sublingual, intranasal, anal, or vaginal, delivery. The treatment mayconsist of a single dose or a plurality of doses over a period of time.

The present invention is further illustrated by the following examples.

EXAMPLES Examples of Heterocomplex Preparations Example 1 Preparation ofMurine KLH-VEGF Heterocomplex

0.58 mg of KLH protein is dissolved in 0.5 ml of 10 mM phosphate buffer,pH 8.5. To this solution is added 1 mg of murine VEGF dissolved in 1 mlof the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature.

The mixture is finally dialyzed under the same conditions as thepreviously performed dialysis.

Example 2 Human KLH-VEGF Heterocomplex

Such a heterocomplex is the active ingredient of a vaccine able tomainly induce in the vaccine the production of antibodies neutralizingthe human VEGF.

0.58 mg of KLH protein is dissolved in 0.5 ml of 10 mM phosphate buffer,pH 8.5. To this solution is added 1 mg of human VEGF dissolved in 1 mlof the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 3 Preparation of Murine KLH-IL4 Heterocomplex

0.841 mg of KLH protein is dissolved in 0,8 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of murine IL4 dissolvedin 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 4 Preparation of a Human KLH-IL4 Heterocomplex

Such a heterocomplex is the active ingredient of a vaccine able tomainly induce in the vaccine the production of antibodies neutralizingthe human IL4.

1 mg of KLH protein is dissolved in 1 ml of 10 mM phosphate buffer, pH8.5. To this solution is added 1 mg of murine IL4 protein dissolved in 1ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 5 Preparation of a KLH-IFNα Complex

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccine the production of antibodies neutralizing thehuman IFNα.

0.625 mg of KLH protein is dissolved in 0,6 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of human IFNα proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 6 Preparation of a gp160-IFNa Complex

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccine the production of antibodies neutralizing both thegp160 structure protein of the HIV-1 virus and the immunosuppressiveIFNα cytokin protein. Moreover, such a heterocomplex should be able toinduce a cell reaction (chemiokins, auxiliary T, CTL) raised against theinfected cells expressing the gp160.

0.380 mg of gp160 protein is dissolved in 0,380 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of human IFNα proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 7 Preparation of a gp160-Toxoid Tat Heterocomplex (The TatProtein is Biochemically Inactivated)

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccine the production of antibodies neutralizing both thegp160 structure protein of the HIV-1 virus and the extracellular Tatprotein of V1H-1. Moreover, such a complex should be able to induce acell reaction (chemiokins, auxiliary T, CTL) raised against the infectedcells expressing the gp160.

0.550 mg of gp120 protein is dissolved in 0.550 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of toxoid Tat proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

Then the reaction is blocked by the addition of final 0.1 M glycine for1 hour at room temperature. The excess glycine is then removed by 3successive 2 hour dialyses each in a dialysis tube with a 3 kDa cutoffthreshold, at 4° C. against 200 ml of phosphate buffer, pH 7.6, 10 mM.

Example 8 Preparation of a gp160-GM Tat Heterocomplex (The Tat Proteinis Genetically Inactivated)

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccinee the production of antibodies neutralizing boththe gp160 structure protein of the HIV-1 virus and the Tat proteinregulating the VIH-l. Moreover, such a heterocomplex should be able toinduce a cell reaction (chemokines, auxiliary T, CTL) raised against theinfected cells expressing the gp160.

0.550 mg of gp160 protein is dissolved in 0.550 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of GM Tat proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

Then the reaction is blocked by the addition of final 0.1M glycine for 1hour at room temperature. The excess glycine is then removed by 3successive 2 hour dialyses each performed in a dialysis tube with acutoff threshold being 3 kDa, at 4° C., against 200 ml of phosphatebuffer, pH 7.6 10 mM.

Example 9 Preparation of Murine KLH-TNFa Heterocomplex

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccine the production of antibodies neutralizing themurine TNFα.

0.625 mg of KLH protein is dissolved in 0.6 ml of 10 mM borate buffer,pH 8.8, 150 mM NaCl. To this solution is added 1 mg of human IFNαprotein dissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 45 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 4-hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM 150 mMNaCl.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 10 Preparation of a Tat Peptide Heterocomplex (19-50)-_(h)IgE

The Tat peptide (19-50) brings auxiliary helper epitopes.

Sequence of the Tat peptide to be used:

-   -   Lys-Thr-Ala-Cys-Thr-Asn-Cys-Tyr-Cys-Lys-Lys-Cys-Cys-Phe-His-Cys-Gln-Val-Cys-Phe-Ile-Thr-Lys-Ala-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccine the formation of (human) anti-IgE antibodies.

0.1 mg of Tat peptide (19-50) (4.06×10⁻⁸ mole) are dissolved in 0.2 mlof 10 mM borate buffer, pH 8.5. To this solution is added 1 mg of humanIgE (6.6×10⁻⁹ mole) dissolved in 1 ml of the same buffer.

The thus prepared mixture is then treated using formaldehyde at thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 2 successive 4 hourdialyses and a final 16 hour dialysis against a large volume ofphosphate buffer, 10 mM, pH 7.4 containing 0.8% of NaCl (PBS), at 4° C.,using a dialysis bag with a cutoff threshold being at 10 kDa. (IgEpeptide ratio=50:1)

Example 11 Preparation of a Heterocomplex Against the Ricin Fragment

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccine the formation of neutralizing antibodies directedagainst the fragment involved in the binding of the ricin molecule,thereby preventing it from exerting its toxic activity.

To 0.5 mg of KLH (1.1×10⁻³ mole) dissolved in 0.5 ml of 10 mM phosphatebuffer, pH 8.2, is added 1 mg (3.3×10⁻⁸ mole) of a fragment of ricindissolved in 1 ml of the same buffer. The thus prepared mixture istreated using formaldehyde at the final concentration of 0.026 M for 30minutes at room temperature.

The excess glutaraldehyde is then removed by 2 successive 4 hour eachdialyses and a 16-hour dialysis against a large volume of phosphatebuffer, 10 mM, containing 0.8% of NaCl (PBS), using a dialysis bag witha cutoff threshold being 3 kDa. (KLH:Ricin-a ratio=1:30).

Examples of Biochemical Characterizations of Heterocomplexes

A. Material and Methods of Examples 12 to 23

The biochemical characterizations of heterocomplexes are performed bymeans of the following techniques:

1. Antigenicity Test

The study of antigenicity of a heterocomplex compared to theantigenicity of proteins making it up is performed by a conventionalindirect ELISA. Such a technique allows for a protein to bequantitatively measured through the specific recognition of an antibodyraised against an antigen. Such a test comprises depositing sampledilutions containing the sought protein in wells of a microtiter plate.A specific polyclonal antibody reacts with the immobilized protein. Asecond antibody, conjugated with horseradish peroxydase, specific to thefirst one is then added. The formed complex is revealed throughincubation with OPD. The resulting yellow color is directly proportionalto the amount of bound proteins. The absorbance (DO) of each well ismeasured by means of a microplate reader. The amount of protein presentin the sample is then determined by means of a calibrating range.

2. Isoelectrofocusing in Agarose Gel Followed by a Western Blot

The isoelectrofocusing in agarose gel allows to separate moleculesdepending on their isoelectric point (Ip) under non denaturingconditions, allowing to study heterocomplexes without destroying theweak bonds existing within such complexes.

The isofocusing is followed by an emergence through a Western blot.After electrophoretic separation, the molecules are transferred on anitrocellulose membrane through capillarity. Such molecules are thencharacterized by immunochemistry.

3. Measurement of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule (First Method)

Estimating the percentage of molecules of the antigen of interestcovalently linked to carrier protein molecules in an immunogenic productoccurs, for example, through molecular sieving on a column containingSuperdex 200, under denaturing (8 M urea) and reducing (5%beta-mercaptoethanol) conditions.

The % of covalently linked molecules of the antigen of interest isdeducted from the amount of antigen of interest (determined by aconventional indirect ELISA) present in the exclusion volume of thecolumn. Indeed, the carrier protein molecule, such as KLH, having amolecular mass much higher than the highest fractionating limit of thecolumn being used (200 kDa in this case), exists in the exclusion volumeof such a column. Thus, under denaturing and reducing conditions, onlythe antigens of interest covalently linked to the carrier proteinmolecule exist in the exclusion volume.

4. Measurement of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule (Second Method)

The percentage of cytokin fixed on the carrier protein (KLH) wasdetermined by a double sandwich ELISA, by means of a capture antibodyspecifically directed against the carrier protein.

100 μl of horse polyclonal antibodies raised against KLH (1 mg/ml)diluted in a 10 mM phosphate buffer, pH 7.3 NaCl 150 mM (PBS) are boundin wells of a microtiter plate (high-binding Costar) for 2 hours at 37°C. After 3 washes in PBS/0.1% Tween 20 (PBST), the wells are saturatedwith PBS containing 2% of BCS.

After 1.30 hour of saturation, the wells are washed three times withPBST, then heterocomplex 2 by 2 dilutions (10, 5, 2.5, 1.25, 0.625,0.312 and 0.156 μg/ml) made in duplicate, are added in the wells (100μl/well).

After 2 hours of incubation, the wells are washed three times with PBST.The Tween, a dissociating agent, present in the washing buffer, allowsto remove all the molecules which are not covalently linked to the KLHbeing, itself, specifically bound on the capture antibody.

Then, both heterocomplex dilutions are treated in two different ways:

a) the first set is incubated with an antibody raised against KLH

b) the second set is incubated with an antibody raised against cytokin.

After 1.30 hour of incubation at 37° C., the wells are washed aspreviously indicated then incubated with a secondary antibody coupled tothe peroxydase, directed against the origin species of the firstantibody. After 1.30 hour of incubation at 37° C., the antibodies arewashed again. Then, the addition of the peroxydase substrate,O-PhenyleneDiamine (OPD) allows for the reevaluation of the presence ofthe KLH bound by the capture antibody and cytokins covalently bound onthe KLH.

The amount of KLH bound by the capture antibody and then the amount ofcytokin molecules covalently bound on the KLH are calculated by means ofcalibrating curves done by ELISA.

The percentage of cytokin covalently bound to the KLH is thendetermined.

Example 12 Biochemical Characterization of the KLH-Murine VEGFHeterocomplex

1. Antigenicity

The KLH-murine VEGF heterocomplex has an antigenicity identical to theantigenicity of murine VEGF.

2. Isoelectrofocusing in Agarose Gel Followed by a Western Blot

FIG. 1 shows that the KLH-murine VEGF heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it

Example 13 Biochemical Characterization of the KLH-Human VEGFHeterocomplex

1. Antigenicity

The KLH-human VEGF heterocomplex has an antigenicity identical to theantigenicity of human VEGF.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 2 shows that the KLH-human VEGF heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it up. The human VEGF sample was deposited at three differentlocations in order to show that it still migrates at the same location.

Example 14 Biochemical Characterization of KLH-Murine IL4 Heterocomplex

1. Antigenicity

The KLH-murine IL4 heterocomplex has an antigenicity identical to theantigenicity of murine IL4.

Example 15 Biochemical Characterization of the KLH-Human IL4Heterocomplex

1. Antigenicity

The human KLH-IL4 complex has an antigenicity equal to the antigenicityof the human IL4 protein.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 3 shows that the human KLH-IL4 heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it up.

Example 16 Biochemical Characterization of the KLH-IFNa Heterocomplex

1. Antigenicity

The human KLH-IFNα complex has an antigenicity identical to theantigenicity of the human IFNα.

2. Estimation of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule

The KLH-IFNα preparation is passed on a superdex S200 column followingthe above described conditions. The apparent peak in the exclusionvolume was collected, dialyzed then freeze-dried. The concentration inantigen of interest was determined by the indirect ELISA technique. TheIFNα amount present in the excluded volume is 30 μg while 1000 μg of IFNwere used for preparing the immunogenic product, without any measurableloss of antigen during the preparing method. The percentage of moleculesof the antigen of interest covalently linked to the KLH molecule in theimmunogenic product comprising KLH-IFNα heterocomplexes can therefore beestimated to approximately 3%.

Example 17 Biochemical Characterization of the gp160-IFNa Heterocomplex

1. Antigenicity

The human gp160-IFNα complex has an antigenicity identical to theantigenicity of the gp160 protein as well as to that of human IFNα.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 4 shows that the human gp160-IFNα heterocomplex migrates under theform of one single strip at a Ip being quite different from the Ip ofthe gp160 protein recombining the component. The Ip of such aheterocomplex is slightly lower than that of IFNα.

Example 18 Biochemical Characterization of the gp160-Toxoid TatHeterocomplex

1. Antigenicity

The gp160-toxoid Tat complex has an antigenicity identical to theantigenicity of the gp160 protein and to that of the Tat protein.

Example 19 Biochemical Characterization of the gp160-GM TatHeterocomplex

1. Antigenicity

The gp160-GM Tat complex has an antigenicity identical to theantigenicity of the gp160 protein and to that of the Tat protein.

Example 20 Biochemical Characterization of the KLH-Murine IL4Heterocomplex

1. Antigenicity

The murine KLH-IL4 heterocomplex has an antigenicity identical to theantigenicity of murine IL4.

2. Estimation of the % of Antigen Molecules of Interest Covalently Boundto the Carrier Protein Molecule

11% of molecules of murine IL4 are covalently fixed to the KLH.

Example 21 Biochemical Characterization of the KLH-IFNα Heterocomplex

1. Antigenicity

The KLH-human IFNα complex has an antigenicity identical to theantigenicity of the human IFNα.

2. Estimation of the % of Antigen Molecules of Interest Covalently Fixedto the Carrier Protein Molecule

8% of molecules of human IFNα are covalently bound to the KLH.

Example 22 Biochemical Characterization of the Tat-_(h)IgE PeptideHeterocomplex

1. Antigenicity

The Tat-_(h)IgE peptide complex has an antigenicity comparable to thatof human IgE.

Example 23 Biochemical Characterization of the KLH-β Ricin-Heterocomplex

1. Antigenicity

The KLH-β Ricin complex has an antigenicity comparable to that of the βricin fragment.

2. Isoelectrofocusing Followed by a Western Blot

The complex migrates under the form of a single strip and the presenceof the P fragment is enhanced by Western Blot.

Examples of Immunogenic Activity of Heterocomplexes Example 24Immunogenic Activity of the KLH-Murine VEGF Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-murine VEGF preparation compared tothat of the murine VEGF was studied in 18 to 20 g BALB c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without any immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is studied in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bycell proliferation test conducted on PBMCs cultivated in the presence ofthe complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice immunized both with the KLH-murine VEGF preparation and themurine VEGF only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine VEGF do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the murine VEGF, determinedby ELISA and expressed in titer (opposite of the dilution giving anoptical density higher than 0.3). FIG. 5 shows the resulting antibodytiters.

The mice immunized with the KLH-murine VEGF preparation show higherantibody titers of the IgG type than those of mice immunized with themurine VEGF only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of VEGF, selective growth factor ofendothelial cells. Endothelial cells (HUVECs) are cultivated in flatbottom wells of a microculture plate at a level of 3,000 cells per well.The sera of each group of mice were pooled. Different dilutions of suchserum pools (1/100-1/800) taken at D-2 and D72 were pre-incubated for 2hours with 20 ng/ml of murine VEGF then deposited on such endothelialcells. The cell culture continued at 37° C. in a humid atmosphere loadedwith 5% of CO₂ for 3 days. 18 hours before the end of the incubation,0.5 μCi of titered thymidine/well were added. The neutralizing seraprevent the murine VEGF from inducing the proliferation of endothelialcells, while non-neutralizing sera allow for the proliferation of suchcells. The results are expressed in neutralization percentage. FIG. 6shows the obtained results.

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the murine VEGF.

Example 25 Immunogenic Activity of the KLH-Human VEGF Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-human VEGF preparation compared tothat of the human VEGF was studied in 18 to 20 g BALB c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1—Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice immunized both with the murine KLH-VEGF preparation and themurine VEGF only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human VEGF do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the human VEGF, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). FIG. 7 shows the resulting antibody titers.

The mice immunized with the KLH-human VEGF preparation show higherantibody titers of the IgG type than those of mice immunized with thehuman VEGF only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of VEGF, selective growth factor ofendothelial cells. Endothelial cells (HUVECs) are cultivated in flatbottom wells of a microculture plate at a level of 3,000 cells per well.The sera of each group of mice were pooled. Different dilutions of suchserum pools (1/100-1/800) taken at D-2 and D72 were pre-incubated for 2hours with 20 ng/ml of human VEGF then deposited on such endothelialcells. The cell culture is continued at 37° C. in a humid atmosphereloaded with 5% of CO₂ for 3 days. 18 hours before the end of theincubation, 0.5 μCi of titered thymidine/well were added. Theneutralizing sera prevent the human VEGF from inducing the proliferationof endothelial cells, while non-neutralizing sera allow for theproliferation of such cells. The results are expressed in neutralizationpercentage. FIG. 8 shows the obtained results.

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human VEGF.

Example 26 Immunogenic Activity of the KLH-Murine IL4 Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the murine KLH-IL4 preparationcompared to that of the murine IL4 was studied in 18 to 20 g BALB cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2 and D72.

3 control mice receive the same preparations without an immunogen.

14 days after the last immunization, the control mice and the miceimmunized with the KLH-murine IL4 were challenged with birch-tree pollenin the presence of alum (100 μg/mice) through the subcutaneous route atD74, D95 and D109. Blood samples are regularly taken in order to followthe occurrence of class G and E antibodies raised against Bet v 1, amajor allergen of the birch-tree pollen.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the KLH-murine IL4 preparation and themurine IL4 only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine IL4 do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the murine VEGF, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). FIG. 9 shows the resulting antibody titers.

The mice immunized with the KLH-murine IL4 preparation show higherantibody titers of the IgG type than those of mice immunized with themurine IL4 only.

The neutralizing activity of those antibodies present in mice immunizedwith the KLH-murine IL4 preparation was measured by means of thebiological activity test of the murine IL4. This test uses HT-2 cells,murine cell lineages the growth of which is IL4 murine-dependent(Watson, J. 1979. J. Exp. Med. 150:1510.). Endothelial cells HT-2 arecultivated in round bottom wells of a microculture plate at a level of10,000 cells per well. Sera diluted at 1/50 taken at D-2 and D72 arepre-incubated for 2 hours with 50 ng/ml of murine H4 then deposited onHT-2 cells. The cell culture is continued at 37° C. in a humidatmosphere loaded with 5% of CO₂ for 3 days. 4 hours before the end ofthe incubation, 0.5 μCi of titered thymidine/well were added. Theneutralizing sera prevent the murine IL4 from inducing the proliferationof HT-2 cells, while non-neutralizing sera allow for the proliferationof such cells. The results are expressed in neutralization percentage.FIG. 10 shows the obtained results.

The antibodies induced by the complex are neutralizing.

Moreover, such neutralizing antibodies raised against murine IL4 preventthe production, by those mice, of antibodies of the IgE type raisedagainst Bet v 1, when the latter are challenged with birch-tree pollen.FIG. 11 indeed shows that mice immunized with KLH-murine IL4 haveneutralizing IgGs raised against murine NL4 blocking the production ofIgE raised against Bet v 1 and start to produce antibodies of the IgGtype directed against Bet v 1. On the other hand, mice which did notreceive any murine KLH-IL4 and therefore not having any antibodies ofthe IgG type directed against IL4, only produce antibodies of the IgEtype directed against Bet v 1.

Example 27 Immunogenic Activity of the KLH-Human IL4 Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-human IL4 preparation compared tothat of the human IL4 was studied in 18 to 20 g BALB c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the KLH-human ILA preparation and the humanIL4 only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IL4 do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IL4, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3).

TABLE 1 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹   <500⁻¹ Control mouse 2 <500⁻¹   <500⁻¹  Control mouse 3 <500⁻¹   <500⁻¹  Miceimmunized with human IL4: mouse 4 <500⁻¹  32,000⁻¹ mouse 5 <500⁻¹ 48,000⁻¹ mouse 6 <500⁻¹  16,000⁻¹ Mice immunized with the KLH-human IL4complex: mouse 7 <500⁻¹ 256,000⁻¹ mouse 8 <500⁻¹ 128,000⁻¹ mouse 9<500⁻¹ 128,000⁻¹

The mice immunized with the KLH-human IL4 preparation show higherantibody titers of the IgG type than those of mice immunized with thehuman IL4 only.

The neutralizing activity of such antibodies induced by the humanKLH-IL4 preparation was measured by means of the biological activitytest of human IL4. This test uses TF-1 cells, human cell lineage thegrowth of which is IL4 human-dependent (Kitamura, T. et al., 1989. J.Cell Physiol. 140:323-34). TF-1 cells are cultivated in round bottomwells of a microculture plate at a level of 10,000 cells per well. Seradiluted at 1/50 taken at D-2 and D72 preincubated for 2 hours with 50ng/ml of human IL4 were then deposited on the TF-1 cells. The cellculture is continued at 37° C. in a humid atmosphere loaded with 5% ofCO₂ for 3 days. 4 hours before the end of the incubation, 0.5 μCi oftitered thymidine/well were added. The neutralizing sera prevent thehuman IL4 from inducing the proliferation of TF-1 cells, whilenon-neutralizing sera allow for proliferation of such cells. The resultsare expressed in neutralization percentage. FIG. 12 shows the obtainedresults.

The antibodies induced by the complex are neutralizing.

Example 28 Immunogenic Activity of the KLH-IFNα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-human IFNα preparationcompared to that of the human IFNα was studied in 18 to 20 g BALB cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1 —Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the KLH-human IFNα preparation and thehuman IFNα only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human NFNA, determinedby ELISA and expressed in titer (opposite of the dilution giving anoptical density higher than 0.3). The table 2 shows the resultingantibody titers.

TABLE 2 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹  <500⁻¹ Control mouse 2 <500⁻¹  <500⁻¹  Control mouse 3 <500⁻¹  <500⁻¹  Miceimmunized with IFNα: mouse 4 <500⁻¹ 96,000⁻¹ mouse 5 <500⁻¹ 128,000⁻¹ mouse 6 <500⁻¹ 96,000⁻¹ Mice immunized with the KHL-IFNα complex: mouse7 <500⁻¹ 96,000⁻¹ mouse 8 <500⁻¹ 96,000⁻¹ mouse 9 <500⁻¹ 128,000⁻¹ 

The mice immunized with the KHL-human IFNα preparation show antibodytiters of the IgG type equivalent to those of mice immunized with thehuman IFNα only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of the human IFNα. (Rubinstein S, J Viral,1981, 755-8). The aim of this test for measuring the antiviral effect isto evaluate the inhibition of the MDBK cell lysis by the VSV (VesicularStomatitis virus) in the presence of IFN. MDBK cells are cultivated inround bottom wells of a microculture plate at a level of 350,000 cellsper well. Different dilutions of sera (1/100 at 1/800) taken at D-2 andD72 were pre-incubated for 2 hours with 5 ng/ml of human IFNa thendeposited on MDBK cells. After 20 hours of cell culture performed at 37°C. in a humid atmosphere loaded with 5% of CO2, the diluted sera presentin the wells are removed, the cells washed, then 100 μl containing 100LD50 (50% lethal dose) of VSV virus are added. 18 hours after theaddition of the virus the lytic effect of the virus is measured. Theneutralizing sera allow the VSV to lyse cells, while non-neutralizingsera prevent such a lysis. The results are expressed in neutralizationpercentage.

TABLE 3 1/100 1/200 1/400 1/800 Mice immunized with IFNα: mouse 4 D-2 00 0 0 D72 100 75 65 50 mouse 5 D-2 0 0 0 0 D72 100 67 60 55 mouse 6 D-20 0 0 0 D72 100 72 65 60 Mice immunized with the KLH-IFNα conjugatemouse 7 D-2 0 0 0 0 D72 100 100 100 100 mouse 8 D-2 0 0 0 0 D72 100 100100 100 mouse 9 D-2 0 0 0 0 D72 100 100 100 100

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human IFNα. The results are expressed inneutralization percentage.

Example 29 Immunogenic Activity of the gp160-IFNα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the human gp160-IFNα preparationcompared to that of the human IFNα was studied in 18 to 20 g BALB cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1—Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice immunized both with the gp160-human IFNa preparation and thehuman IFNα only, do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-human IFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IFN, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3).

TABLE 4 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹  <500⁻¹ Control mouse 2 <500⁻¹  <500⁻¹  Control mouse 3 <500⁻¹  <500⁻¹  Miceimmunized with IFNα: mouse 4 <500⁻¹ 64,000⁻¹ mouse 5 <500⁻¹ 96,000⁻¹mouse 6 <500⁻¹ 128,000⁻¹  Mice immunized with the gp160-IFNα complex:mouse 7 <500⁻¹ 96,000⁻¹ mouse 8 <500⁻¹ 96,000⁻¹ mouse 9 <500⁻¹ 64,000⁻¹

The mice immunized with the gp 160-human IFNa preparation present IgGtype antibody titers equivalent to those of mice immunized with thehuman IFNa only.

The neutralizing activity of such antibodies has been measured with thehelp of the human IFNa biological activity test described in the formerexample. Results are given in neutralization %.

TABLE 5 1/100 1/200 1/400 1/800 Mice immunized with the IFNα: mouse 4D-2 0 0 0 0 D72 100 80 70 53 mouse 5 D-2 0 0 0 0 D72 100 70 65 50 mouse6 D-2 0 0 0 0 D72 100 65 60 57 Mice immunized with the gp160-IFNαconjugate: mouse 7 D-2 0 0 0 0 D72 100 100 100 100 mouse 8 D-2 0 0 0 0D72 100 100 100 100 mouse 9 D-2 0 0 0 0 D72 100 100 100 100

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human IFNα.

Example 30 Immunogenic Activity of the gp160-Toxoid Tat Heterocomplex

A. Material and Methods

The immunogenic (humoral and cellular) activity of the gp160-toxoid Tatpreparation compared to that of the toxoid Tat was studied in 18 to 20 gBALB c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D-2.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results:

1—Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice immunized both with the gp160-toxoid Tat preparation and thetoxoid Tat only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-toxoid Tat do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the Tat, determined by ELISAand expressed in titer (reciprocal of the dilution giving an opticaldensity higher than 0.3). Table 6 shows the resulting antibody titers.

TABLE 6 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹  <500⁻¹ Control mouse 2 <500⁻¹  <500⁻¹  Control mouse 3 <500⁻¹  <500⁻¹  Miceimmunized with toxoid Tat: mouse 4 <500⁻¹ 48,000⁻¹ mouse 5 <500⁻¹64,000⁻¹ mouse 6 <500⁻¹ 48,000⁻¹ Mice immunized with gp160-toxoid Tatconjugate: mouse 7 <500⁻¹ 64,000⁻¹ mouse 8 <500⁻¹ 128,000⁻¹  mouse 9<500⁻¹ 64,000⁻¹

The mice immunized with the gp160-toxoid Tat preparation show higherantibody titers of the anti-Tat IgG type than those of mice immunizedwith the toxoid Tat only.

The neutralizing activity of such antibodies was measured by means ofthe Cat assay. Different dilutions of sera (1/100-1/800) taken at D-2and D72 are incubated for 2 hours with 50 ng/ml of native Tat. Suchdilutions are then deposited on HeLa cells, stably infected cells with aplasmid containing LTR of the VIH-1 as the promotor of theChloramphenicol Acetyl transferase gene (CAT). After 24 hours ofculture, the cells are lysed and the amount of CAT protein produced ismeasured by an ELISA test, the Cat assay (Boehringer Mannheim).Neutralizing sera prevent the Tat protein from inducing the expressionof the CAT protein, while the non-neutralizing sera allow for thesynthesis of such CAT protein. The results are expressed inneutralization %.

TABLE 7 1/100 1/200 1/400 1/800 Mice immunized with toxoid Tat: mouse 4D-2 0 0 0 0 D72 60 50 25 20 mouse 5 D-2 0 0 0 0 D72 60 55 30 20 mouse 6D-2 0 0 0 0 D72 65 50 30 30 Mice immunized with gp16-toxoid Tatconjugate: mouse 7 D-2 0 0 0 0 D72 100 100 100 100 mouse 8 D-2 0 0 0 0D72 100 100 100 100 mouse 9 D-2 0 0 0 0 D72 100 100 100 100

The antibodies induced by the gp160-toxoid Tax conjugate have a higherneutralizing power than that induced by the toxoid Tat

2. Production of MIP1α

The production of MIP1α in culture supernatants of splenocytes.Splenocytes of immunized mice and control mice are isolated thencultivated in round bottom wells of a micro-culture plate at a level of100,000 cells/well in the presence of 5 Mg/ml of p24, gp160, native Tatand a mixture of 5 μg/ml gp160 and 5 μg/ml of native Tat. Thesupernatants are taken after 24 hours of culture and the presence ofMIP1 in the supernatants is measured by means of a R&D ELISA test. Theresults are expressed in μg/ml.

TABLE 8 Gp160 + Gp160 native Tat native Tat P24 Control mice: mouse 1MIP1α 95 90 145 9 D72 mouse 2 Mip1α 100 90 136 7 D72 mouse 3 MIP1α 120110 132 9 D72 Mice immunized with toxoid Tat: mouse 4 MIP1α 145 130 1907 D72 mouse 5 MIP1α 128 145 225 9 D72 mouse 6 MIP1α 150 230 295 10 D72Mice immunized with gp160-toxoid Tat conjugate: mouse 7 MIP1α 875 7361725 9 D72 mouse 8 MIP1αα 945 905 1900 7 D72 mouse 9 MIP1α 1025 795 17558 D72

Splenocytes of mice immunized with the gp160-toxoid Tat conjugateproduce more MIP1α chemiokins than cells of mice immunized by the toxoidTat only when they are activated, in vitro, by the immunogens usedduring the immunization.

4. Proliferation of Splenocytes of Immunized Mice (CMI test)

Splenocytes of immunized mice and of control mice are isolated thencultivated in round bottom wells of a micro-culture plate at a level of100,000 cells/well in the presence of p24, gp160, native Tat and amixture of gp160 and native Tat. The cell culture is continued at 37° C.in a humid atmosphere loaded with 5% of CO2 for 6 days. 18 hours beforethe end of the incubation, 0.5 μCi of titered thymidine/well were added.The intensity of the immune response is proportional to theproliferation index Ip.

Ip=spm (strokes per minute) for the given antigen/control spm

TABLE 9 Gp160 + native Gp160 native Tat Tat P24 Control mice: mouse 11.1 1.1 1 1.2 D72 mouse 2 1 1.1 1.1 1.1 D72 mouse 3 1.2 1 1 1.1 D72 Miceimmunized with toxoid Tat: mouse 4 1.2 8 10 1.1 D72 mouse 5 1 9 9 1.2D72 mouse 6 1 10 9 1.2 D72 Mice immunized with gp160-toxoid Tatconjugate: mouse 7 9 11 8 1 D72 mouse 8 10 9 7.5 1 D72 mouse 9 10.5 9 81 D72

Splenocytes of mice immunized with the gp160-toxoid Tat conjugate or thetoxoid Tat, proliferate, when they are activated, in vitro, with theimmunogens used during the immunization.

Example 31 Immunogenic Activity of the gp160-GMTat Heterocomplex

A. Material and Methods

The immunogenic (humoral and cellular) activity of the gp160-GM Tatpreparation compared to that of the toxoid Tat was studied in 18 to 20 gBALB c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an emulsion in AIF through the intramuscular route. A 5 μgbooster injection in AIF is given at D-2.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice immunized both with the gp160-GM Tat preparation and the toxoidTat only, do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-toxoid Tat do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the Tat, determined by ELISAand expressed in titer (reciprocal of the dilution giving an opticaldensity higher than 0.3). Table 10 shows the resulting antibody titers.

TABLE 10 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹  <500⁻¹ Control mouse 2 <500⁻¹  <500⁻¹  Control mouse 3 <500⁻¹  <500⁻¹  Miceimmunized with GM Tat: mouse 4 <500⁻¹ 64,000⁻¹  mouse 5 <500⁻¹ 64,000⁻¹mouse 6 <500⁻¹ 48,000⁻¹ Mice immunized with the gp160-GM Tat conjugate:mouse 7 <500⁻¹ 128,000⁻¹  mouse 8 <500⁻¹ 128,000⁻¹  mouse 9 <500⁻¹64,000⁻¹

The mice immunized with the gp160-GM Tat preparation show higherantibody titers of the anti-Tat IgG type than those of mice immunizedwith the GM Tat only.

Example 32 Immunogenic Activity of the KLH-Murine TNFα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-murine TNFα preparationcompared to that of the murine TNFα was studied in 18 to 20 g BALB/cmouse.

At day 0, a group of 3 mice (group A) receives a 0.1 ml injection of anAIF emulsion through the intramuscular route containing 60 μg of theKLH-TNFa complex. A booster injection of 30 μg and 15 μg in AIF is givenrespectively at D21 and D60. 3 control mice receive a dose equivalent inmurine TNFα according to the same protocol. (group B)

At day 0, a group of 3 mice (group C) receives a 0.1 ml injection in AIFthrough intramuscular route containing 60 μg of KLH-murine TNFαheterocomplex and 30 μg of the phosphorothioate oligodeoxynucleotide5′-TCCATGACGTTCCTGACGTT-3′ (CpG ADN: 1826). A booster injection of 30 μgand of 15 μg of the KKL-murine TNFa heterocomplex in AIF is givenrespectively at D21 and D60. 3 control mice receive a dose equivalent inmurine TNFα according to the same protocol. (group D)

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the murine KLH-TNFα preparation and themurine TNFa only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine TFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex with orwithout the DNA CPG 1826 show any sign of toxicity (temperature,cutaneous disorders, systemic or regional signs) during the 7 daysfollowing the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the murine TNFα, determined byELISA and expressed in titer. The presence of antibodies of the IgA typedirected against the murine TNFa in vaginal secretions was alsodetermined by ELISA and expressed in titer. The titer represents theopposite of the dilution giving an optical density higher than 0.3. Thefollowing table shows the resulting antibody titers.

TABLE 11 Vaginal IgA D-2 D72 D-2 D72 Mice immunized with KLH-murine TNFα(group A) 1 <500⁻¹ 64,000⁻¹ <10⁻¹ 20⁻¹ 2 48,000⁻¹ 20⁻¹ 3 64,000⁻¹ 40⁻¹Mice immunized with the Murine TNFα (group B) 4 <500⁻¹   750⁻¹ <10⁻¹10⁻¹ 5  1,000⁻¹ 20⁻¹ 6   750⁻¹ 10⁻¹ Mice immunized with KLH-murine TNFαin the presence of CPG (group C) 7 <500⁻¹ 128,000⁻¹  <10⁻¹ 160⁻¹  8256,000⁻¹  80⁻¹ 9 256,000⁻¹  320⁻¹  Mice immunized with murine TNFα: inthe presence of CpG (group D) 7 <500⁻¹   2000⁻¹ <10⁻¹ 20⁻¹ 8  4,000⁻¹40⁻¹ 9  3,000⁻¹ 40⁻¹

Example 33 Immunogenic Activity of the Tat Peptide-_(h)IgE Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-human IgE preparationcompared to that of the human IgE was studied in 18 to 20 g BALB cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

2—Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the KLH-human IgE preparation and the humanIgE only, do not show any clinical sign and no anatomic wound. Theimmunosupression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IgE do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IgE, determined byELISA and expressed in titer (reciprocal of the dilution giving anoptical density higher than 0.3). The following table shows theresulting antibody titers.

TABLE 12 D-2 D72 Control mice: 1 <500⁻¹   <500⁻¹  2 3 Mice immunizedwith _(h)IgE 4 <500⁻¹  64,000⁻¹ 5 128,000⁻¹ 6 128,000⁻¹ Mice immunizedwith KLH-_(h)IgE: 7 <500⁻¹ 256,000⁻¹ 8 128,000⁻¹ 9 256,000⁻¹

The mice immunized with the KHL-_(h)IgF preparation show antibody titersof the IgG type slightly higher than those of mice immunized with thehigE preparation only.

Example 34 Immunogenic Activity of the KLH-Ricin-β Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-Ricin-β preparationcompared to that of the ricin β fragment was studied in 18 to 20 gBALB/c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

3—Lack of Toxicity of the Heterocomplex in Vivo and in Vitro

The mice both immunized with the human KLH-Ricin-preparation and thericin β-fragment only do not show any clinical sign and no anatomicwound. The immunosuppression test shows that doses of 100 ng/ml to 1μg/ml of KLH-ricin β do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the β fragment of ricin,determined by ELISA and expressed in titer (reciprocal of the dilutiongiving an optical density higher than 0.3). The following table showsthe resulting antibody titers.

TABLE 13 Titer D-2 D72 Control mice: 1 <500⁻¹   <500⁻¹  2 3 Miceimmunized with ricin-β 4 <500⁻¹ 256,000⁻¹ 5 512,000⁻¹ 6 256,000⁻¹ Miceimmunized with KLH-Ricin-β 7 <500⁻¹ 256,000⁻¹ 8 256,000⁻¹ 9 128,000⁻¹

The neutralizing activity of such antibodies was checked by theinjection f mixtures of anti-Ricin-β and ricin serum which did not causethe animal's y to what was observed during the administration to themouse of ricin and mixtures.

Example 35 Method of Manufacturing a Stable Immunogenic ProductAccording to the Invention

A. Materials

TABLE 14 Chemicals and Reagents Reagent Grade Supplier DetailsApplication Glutaraldehyde Grade 1 Sigma G5882 10 × 1 mL PD & GMP 25%Formaldehyde — Sigma F-1635 25 mL PD 37% Formaldehyde EP Sigma 15513 GMP37% Di-sodium Analar BDH 10249 PD hydrogen phosphate (anhydrous)Di-sodium USP Merck 1.06585 5 kg GMP hydrogen phosphate (anhydrous) EDTAAnalar BDH 10093 PD EDTA USP Merck 108421 1 kg GMP Glycine Analar BDH10119 PD Glycine USP Merck 500190 1 kg GMP Sodium chloride Analar BDH10241 PD Sodium chloride BP Merck 116224 GMP DMSO 99.5% Sigma D5879 PDDMSO USP Sigma D2438 10 mL GMP DPBS — Sigma D8537 PD DPBS tbc tbc tbcGMP

TABLE 15 Consumables Description Supplier Application Slide-a-Lyzer ®cassettes Perbio; 66370 Dialysis 7 kDa MWCO (0.5-3 mL) Pellicon XL PESMillipore; PXB010A50 Tangential flow membranes (5 or 10 kDa filtrationMWCO) 50 mL tubes Nunc; 362696 Sample container Wide mouth polypropyleneVWR; 215-0520 Sample container bottle (150 mL) Wide mouth polypropyleneVWR; 215-5683 Sample container bottle (250 mL) Cryovials (3.5 mL & 2 mL)Sigma; V1138; V9637 Sample container Labscale TFF unit with Millipore;XX42LSS13 Tangential flow 500 mL acrylic reservoir filtration

B. Description of the Method

Step a)

-   -   Take 30.05 mL of TNFa (at 4.2 mg/ml) and allow to thaw at 4° C.        overnight (NB: 126.21 mg of TNFa required).

Step b) (or step b1) in Certain Embodiments of the Method)

-   -   Add 90.15 mL of “dilution buffer” to obtain the “working buffer”        solution and TNFa at 1 mg/mL (±10%).        -   Dilution buffer=130 mM di-sodium hydrogen phosphate, 133 mM            NaCl, 6.6 mM EDTA, pH 7.8.        -   Working buffer=100 mM di-sodium hydrogen phosphate, 150 mM            NaCl, 5 mM EDTA, pH 7.8.

Step b2)

-   -   To the remaining 120 mL of the TNFa at 1 mg/mL (±10%) and add        1.2 mL of DMSO. Hold the mixture at RT for 30 minutes. Mix by        agitation every 10 minutes and attempt to avoid foam formation.    -   Add 61.34 mL of “working buffer” to the mixture. Mix gently by        inverting the container and attempt to avoid foam formation.

Step c)

-   -   Add 51.6 mg of KLH. Note: for a 9.81 mg/mL solution of KLH add        5.26 mL. Mix gently by inverting the container. The total volume        at this point is 187.8 mL.

Step d)

-   -   Dilute the 25% stock of glutaraldehyde to 2.5% using the        “working buffer”. This is done immediately prior to use.    -   Add 20.86 mL of the diluted 2.5% solution of glutaraldehyde.        Note that the total volume at this point is 208.56 mL. Mix        gently by inverting the container.    -   Close the bottle and incubate for 45 min at RT. Gentle turn the        bottle over every 15 min.

Step e)

-   -   Diafilter the solution using TFF against the working buffer    -   dialysis three times against 20 volumes of phosphate buffer at        pH 7.6 10 mM, 150 mM NaCl.

Volume 1=2 hours

Volume 2=2 hours

Volume 3=overnight

Step f)

-   -   Dilute stock solution of formaldehyde (at 37%) 10-fold with        “working buffer” to give a 3.7% solution. This is done        immediately prior to use.    -   Add diluted 3.7% formaldehyde to the TNFa-KLH solution to give a        final concentration of 0.2%.

For example, if the volume recovered is 200 mL, then the amount of 3.7%formaldehyde to be added will be 11.43 mL).

-   -   Seal the bottle and incubate for 6 days at 37° C. The bottle is        overturned once a day.

Step g)—Addition of Glycine

-   -   After the 6 days, add 2M glycine (made up in WFI) to a final        concentration of 0.1M). Incubate for 1 hr at room temperature        during which time period the bottle is gently overturned.

Step h)

-   -   Check the pH of the DPBS (Sigma) to be used for diafiltration        and adjust if required to a final pH of 7.3±0.2 using 0.1M NaOH.        No need for pH adjustment is normally expected.    -   Diafilter the solution using TFF against DPBS.

2×5 kDa MWCO TFF membranes will be used

A 500 mL (acrylic) reservoir on the Labscale TFF system will be used.

Material is recovered from the membrane by draining the concentrate(ultrafiltration retentate) into a pre-weight polypropylene bottle.

The process disclosed in this example illustrates an embodiment of themethod according to the invention for manufacturing a stable immunogenicproduct comprising antigenic heterocomplexes of TNFa and KLH.

Further, the method which is detailed hereunder is designed formanufacturing a batch of 120 mg of the stable immunogenic product.

Example 36 Test Procedures for the Stable Immunogenic Product Obtainedby the Method According to the Invention

The test procedures disclosed in Example 36 may be referred to by oneskilled in the art. However, the one skilled in the art may also performtest procedures according to any one of the test procedures that aredisclosed in the examples 37 to 40 herein.

2.1. Total Protein Content Determination by Colorimetry: Example of theBradford Test

The protein content is determined using the Bradford technique(Bradford, M. Anal. Biochem. 1976.72, 248-254).

Briefly, a calibration curve is established with bovine serum albumin(BSA) by pipetting in a series of test tubes 0, 10, 20, 30, 40 μL of a0.2 mg/mL BSA solution in PBS. Subsequently, the volume of each tube iscompleted to 500 μL by adding the corresponding volume of DI water. Toeach tube is then added 500 μL of Bradford reagent. Two blanks areprepared with 200 μL PBS. After 5 min. reaction at room temperature, thecontent of each tube is vortexed and read at 595 nm.

Two hundred μL of an appropriate dilution of the test protein solutionare reacted with the Bradford reagent as described above.

TNFa Protein Content in KLH-TNFa: ELISA

Four samples of KLH-TNFa taken from 4 vials are diluted in PhosphateBuffered Saline pH 7.2 (PBS) at 5 μg/mL. These dilutions are used forcoating microtiter plates (Costar 3590), 100 μL per well. After allowingcoating to occur overnight at 4° C., the plates are washed with PBScontaining 0.1% Tween 20 and the wells are saturated with 2% Fetal CalfSerum (FCS) solution in 100 μL PBS-Tween for 2 hr at 37° C. Afterwashing the plates with PBS-Tween, 100 μL of serial dilutions of ananti-TNFa serum are pipetted into each well and the plates are incubatedfor 2 hr at 37° C., after which they are thoroughly washed and againincubated for 2 hr at 37° C., after adding to each well 100 μL of aHRP-labeled anti-IgG antiserum.

Following incubation, the plates are washed and 100 μL ofortho-phenylenediamine (OPD) solution are added to each well. Threeminutes later, 100 μL of 2N sulphuric acid are added to each well andthe plates are read at 490 nm in a multiscan photometer.

2.2. Purity Grade of KLH-TNFa Immunogen: IEF+Western Blot

The purity grade of the immunogen is assessed by isoelectric focusing(IEF) by allowing the KLH-TNFa immunogen to migrate aside KLH alone andTNFa alone on a 1% agarose plate in a 3 to 10 pH gradient. Afterapplying an appropriate dilution of each sample at 250 μg/mL, themigration is carried out using a Phast system apparatus (AmershamPharmacia) under the following conditions:

1st step (pre-migration): 500 V, 2.5 mA, 2.5 W, 15° C., 5 aVh

2^(nd) step (application): 200 V, 2.5 mA, 2.5 W, 15° C., 5 aVh

3^(rd) step (migration): 1500 V, 2.5 mA, 2.5 W, 15° C., 450 aVh

Following migration, the proteins are transferred by micro-capillarityto a PVDF membrane and the proteins are revealed by Western Blot.

Western Blot Detection

The nitrocellulose membrane is saturated by dipping it overnight in 5%milk-TBS Tween 20 at room temperature, after which it is incubated for 1hr with either the primary polyclonal anti-TNFa (hu) or anti-KLHantibody diluted in 10% milk-TBS Tween 20 at room temperature.Subsequently, the membrane is washed 4 times during 5 min with TBS-Tween20 before being incubated for 1 hr with the secondary HRP-labeledantibody diluted with 10% milk-TBS Tween 20 at room temperature. Again,the membrane is washed 4 times with TBS-Tween 20 and the spots arerevealed by chemo-luminescence using an ECL Plus Kit (AmershamPharmacia).

3. Percentage of CYT-KLH Covalent Bonds in a CYT Kinoid

3.1. Method No 1: Size Exclusion Chromatography in Denaturing andReducing Conditions.

The test kinoid solution is submitted to denaturing (urea 8M finalconcentration) and reducing (beta-mercaptoethanol 5% finalconcentration) conditions that will lead to a dissociation ofnoncovalent bonds. The resulting solution is eluted through asize-exclusion column packed with Superdex 200™ (Pharmacia), chosen forits 200 kDa fraction limit, far below the molecular weight of KLH andcovalent KLH-TNFa constructs. Only those cytokine molecules covalentlylinked to KLH will be present in the exclusion volume. The latter isassayed for TNFa (specific TNFa antigens) using a sandwich-ELISAtechnique with anti-TNFa Abs that have no cross-immune reaction withKLH. The result is expressed as a percentage of TNFa titer in thestarting solution, which is measured by the same ELISA technique. Thispercentage is equal to the % TNFa-KLH covalent bonds in the TNFa kinoid.

3.2 Method No 2: Double-Sandwich ELISA with Tween Washing

A 1 mg/mL solution of anti-KLH polyclonal Abs in PBS (10 mM pH 7.3 NaCl150 mM) is used for coating a microtiter plate (Costar, high-binding),100 μL per well, for 2 hr at 37° C. After 3 washing cycles with PBST(PBS with Tween 20 0.1% v/v), the wells are saturated for 1 h30 with PBScontaining 2% FCS. The wells are again washed 3 times with PBST.

Two identical series of dilutions of the test kinoid (10, 5, . . . ,0.156 μg/mL) are then added in the wells and incubated for 2 hr. Thewells are washed 3 times with PBST, the dissociation action of which(via Tween 20) eliminates all molecules non-covalently bound to KLH, thelatter KLH being fixed to the support-coated capture Abs. The firstseries of test dilutions is incubated with anti-KLH Abs while the secondseries is incubated with anti-TNFa Abs. After incubating for 1 h30 at37° C., wells are washed as described above, then incubated withspecific peroxidase-coupled secondary Abs.

The addition of OPD, the peroxidase substrate, allows a quantitativecalorimetric detection of the fixed anti-TNFa Abs, for the first series,and fixed anti-KLH Abs, for the second series. The ratio of fixed Absbetween the two series gives the percentage of TNFa covalently bound toKLH in the kinoid.

4. Immunological Activity of KLH-TNFa Kinoids

Antigenicity Test: ELISA

This test is intended for measuring the capacity of an immunogen (eitheran antigen derivative, or an antigen bound to a carrier protein) tocombine with a specific antibody with respect to that of the nativeantigen. This test essentially consists in a reverse ELISA.

A series of increasing dilutions of the immunogen under test aredistributed into wells of a polystyrene microtiter plate. A specificpolyclonal antibody directed to the protein to be tested is allowed toreact with the immunogen immobilized in the wells. After removing theexcess of antibody unreacted by washing the plate, the Ab immobilized bythe immunogen in the wells is quantitatively revealed by having itreacted with a HRP-labeled Ab directed to the first Ab, the yellow colorthat develops by adding an appropriate substrate is directlyproportional to the amount of immobilized protein. The optical density(OD) of each well is measured with an absorbance microplate reader. Theprotein content in the test sample is determined from a calibrationcurve.

5. Immunogenicity of Kinoids

The immunogenicity of an immunogenic preparation is:

-   -   1—its capacity level to induce the formation of specific        anti-TNFa polyclonal Abs (measured in vivo); and    -   2—the neutralization capacity thereof. The latter is measured in        vitro by quantifying the capacity of the antiserum, sampled from        the immunized mice, to inhibit the specific biological activity        of TNFa    -   1—For this, groups of 20 7-week-old BALB/c mice, 18-20 g body        weight, housed in separate cages of 5 animals, fed with standard        diet in pellets with food and water ad libitum are immunized on        day 0, 7, 14 and 21 by intramuscular administration of 0.1 mL        (10 μg) of the test immunogen in ISA 51 (1:1 v/v emulsion in ISA        51). A booster injection of 5 μg of the immunogen is given as        0.1 ml of a 1:1 emulsion in ISA 51 on day 60. A blood sample is        taken 2 days before initiating the immunization by retro-orbital        puncture and again 12 days after the last injection for antibody        level determination by ELISA. Three control mice receive a 1:1        v/v emulsion of PBS in IFA.

Serum Ab titers are expressed as the inverse of the dilution that givesan OD>0.3.

-   -   2—Neutralization capacity: [ACOMPLETER]

The results show the high neutralizing capacity of the sera antibodiesof mice immunized with a stable immunogenic product of the inventioncomprising TNFa and KLH.

6. Immunotoxicity—In Vitro

Test of proliferation (by incorporation of 3H-thymidine) of T cellsstimulated by PPD/TT antigens and treated with various kinoid doses.

PBMC's are freshly isolated from healthy donors by blood separation byFicoll gradient centrifugation. Cells are introduced in round-bottom96-well plates, 15,000 cells/well in RPMIc medium with 10% FCS. Thekinoid is added at concentration 1 μg/mL to 100 ng/mL, then PPD or TT at0.16%. Plates are allowed to incubate at 37° C. 5% CO2 for 6 days.Tritiated thymidine is added 18 hr prior to incubation end, 0.5μCi/well. Cell proliferation is analyzed with a β scintillation counter.

7. Biological Characteristics of KLH-TNFa Kinoids TNF-α Bio-Assay:

Cytolysis of Murine L929 Cells in the Presence of Actinomycin D

Materials

-   -   L929 mouse fibroblast line (ATCC Cat. No. CCL-1) KLH-TNFα        (murine or human) kinoid in PBS (standard), NEOVACS    -   Recombinant murine TNFα Peprotech (315-01A) or human TNFα        Peprotech (300-01A) Culture Medium (RPMI supplemented with 10%        FCS (Foetal Calf Serum) 2 mM glutamine, 100 U/ml        penicillin-streptomycin    -   Assay Medium (RPMI supplemented with 2% FCS 2 mM glutamine, 100        U/ml penicillin-streptomycin)    -   Pre-incubation medium: HL1 supplemented with 2 mM glutamine, 100        U/ml penicillin-streptomycin    -   96-well flat-bottom culture plate (Costar, 3595)    -   Actinomycin D, 1000 μg/mL stored at 4° C. (protected from light)    -   MTT solution (Sigma, M5655) 5 mg/mL stock in PBS stock aliquot        kept at minus −20° C. (protected from light)    -   DMSO (SIGMA, 471267)

Experiment Duration

24-hour incubation

1 hour assay preparation

Method

1. Dilute KLH-TNFα murine or human kinoid and standard in a series oftwo-fold dilutions in the Assay Medium in 50 μL/well in 96-wells platefrom row 2 to 11, starting at thr proper dilution Leave row 1 as blank.

2. Prepare L929 cell suspension at a density of 7.5 105/mL in AssayMedium supplemented with actinomycin D at 1 μg/mL (protected fromlight). Add 50 μl/well of the cell suspension to the same plate, fromrow 1 to 12.

3. Incubate plate for 24 h at 37° C. 5% CO2 in a humidified incubator.

4. Rinse 2 times with PBS without Ca2+ Mg2+

5. Add 50 μl/well MTT solution at 40% in Assay Medium and incubate for 4hours at 37° C. 5% CO2 in a humidified incubator.

6. Empty plates and add 50 μL of DMSO to each well.

7. Read plate at 550-630 nm

8. Analyze data

Example 37 Comparative Study of the Various Preparation of KLH-TNFα(Human) Kinoids

Various preparation of a human TNF-α kinoid consisting of human TNF-αcomplexed to the specific carrier protein KLH have been performed.

More precisely, various preparations of KLH-TNFα (human) have beenproduced by the same general process that is disclosed in example 35 butwith variations in (i) the percentage of DMSO, (ii) glutaraldehydeconcentration (2.5 or 22.5 mM) as well as (iii) the time period ofincubation of the intermediate product with formaldehyde (from 0.2 to 6days incubation time period).

Then, the loss of the biological activity of the human TNF-α has beenassayed. Also, the preservation of the conformational B-epitopes in thefinal kinoid product has been tested through (i) the cytotoxicity assayof human TNFα on the L929 cell line, as well as (ii) through theimmunogenicity assays disclosed herein in this example.

A. Materials and Methods

A.1 Assays for the Absence of TNFα Biological Activity in the KLH-TNFα(Human) Kinoids.

Human TNFα, in the presence of D actinomycin, possesses a cytotoxicactivity on the murine fibroblast cells of the L929 cell line, whichcytotoxic activity is assessed by the cell viability test usingMTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide. Thisassay is based on the MTT reduction by the mitochondrial NADPH reductaseof the living cells towards the reduced product formazan which has apurple-blue color.

In this assay, the metabolic activity of the cells allows the evaluationof their viability.

The assessment of the biological activity of the KLH TNF-α kinoidconsists of incubating the L929 cells in the presence of decreasingamounts of said kinoid product (50 ng/ml to 0 ng/ml) in combination withD actynomycin (1 μg/ml) in flat-bottomed wells of a microculture plate.

The cell culture is then performed at 37° C. in a wet atmospherecontaining 5% CO2 during a time period of 24 hours.

After a 24 hours culture time period, the cell culture supernatants arediscarded and replaced by the MTT assay solution.

After 4 hours time period incubation at 37° C. with MTT, MTT solution isremoved and DMSO is added.

After homogenization, the optical density of the cell culturesupernatant is analyzed with a spectrophotometer at a wavelength of 570nm.

The results are expressed as optical density (O.D.) at 570 nm. Inparallel, cell cultures incubated with a range of TNFα from 0 to 10ng/ml is performed, as a control.

The final results are expressed as lethal dose 50, also termed LD50,which is the dose that induces the lysis of 50% of the cultured L929cells.

A.2 Assay for the Preservation of the Conformational B-Epitopes of HumanTNFα in the Final KLH-TNFα (Human) Kinoid, After the Chemical Treatmentswith Glutaraldehyde and Formaldehyde: Immunogenicity Assay.

The study of the preservation of the conformational of B epitopes of thehuman TNFα in the final KLH-TNFα (human) kinoid, after the chemicaltreatments, is performed by a test of the immunogenic activity inC57Bl/6 weighing 18-20 g.

At Day 0, a group of tree mice is injected with 0.2 ml (50 μg) of anemulsion in Complete Freund Adjuvant (CFA) by the intramuscular route.

A second injection of 25 μg of the kinoid product in incomplete Freundadjuvant is administered at Day 21.

Retro-orbital blood sampling is performed on each mouse before the firstinjection and also at Day 28. Sera from each group of mice are gathered.

The humoral response is measured through detection of IgG isotypeantibodies directed against human-TNFα in the sera of the immunizedmice. The humoral response is determined by an ELISA assay and isexpressed in antibody titers (dilution-1 giving an optical densitygreater than 0.3).

The neutralizing capacity of the sera from mice immunized with theKLH-TNFα (human) kinoid has been measured through the TNFα cytotoxicityassay on L929 cells, as described hereafter.

L929 cells are treated with various dilutions from pools of sera(dilutions from 1/100 to 1/12800) that have been sampled at Day −2 andDay 28 and then have been incubated during 40 minutes at roomtemperature, and then 20 minutes at 4° C. with 20 ng/ml of human TNFα.

The cell culture is pursued at 37° C. in wet atmosphere, 5% CO2, during24 hours.

After a 24 hours cell culture time period, the cell culture supernatantsare removed and replaced by MTT solution.

After a 4-hour incubation at 37° C., MTT is removed and DMSO is added.

After homogenization, the optical density of the cell culturesupernatant is analyzed with a spectrophotometer at the wavelength of570 nm.

The results are expressed as optical density (O.D.) of the cell culturesupernatants. A cell cultures incubated with a range of TNFα from 0 to10 ng/ml is performed in parallel, as a control.

The sera that neutralize the cytotoxic activity of TNFα prevent to exertits cytotoxic activity on the L929 cells.

The results are expressed as the neutralizing capacity 50%, or NC50,which corresponds to the sera dilution that neutralizes 50% of thecytotoxic activity of the human TNFα.

B: Results

Table 16 hereunder discloses the results obtained with the final KLHTNFα (human) kinoid prepared according to various chemical treatments.

TABLE 16 Glutaraldehyde Formaldehyde DMSO LD₅₀ Antibody Code (mM) (day)EDTA (%) (ng/ml) titer NC₅₀ K1 2.5 2 5 mM 0 12.5 >64000 1/650 K2 2.5 2 5mM 2 20 >64000 1/600 K 3 22.5 0 5 mM 1 0.15 >64000 1/600 K4 22.5 2 5 mM1 200 >64000 1/750 K5 22.5 2 5 mM 2 225 >64000 1/400 K6 22.5 2 5 mM 5250 >64000 1/700 K7 22.5 6 5 mM 1 >400 >64000 1/1200 K8 22.5 6 5 mM2 >400 >64000 1/300 K9 22.5 6 5 mM 5 >400 >64000 1/350 K10 22.5 2 0 mM 015 >64000 1/700

As shown in Table 16 above, the best results are obtained with a processof manufacturing KLH TNFα (human) kinoid involving a chemical treatment(i) with DMSO at the concentration of 1% and (ii) with glutaraldehyde atthe concentration of 22.5 mM, then (iii) with a chemical treatment withformaldehyde during the period of time of 6 days. The resulting finalKLH-TNFα (human) product is devoid of the cytotoxicity activity of humanTNFα and induces the production of polyclonal antibodies that neutralizethe biological activity of the native human TNFα.

Thus, the manufacturing process according to the condition disclosed inK7 of table 16 above allows the inactivation of the human TNFα cytokinecontained therein why preserving the conformational B-epitopes.

The KLH-TNFα (human) final product prepared according to the conditionsnamed “k7” in Table 16 above is the same as the one which ismanufactured according to example 35 above.

This best TNFα kinoid product is the one which has been studied in thefollowing examples, notably in a transgenic mice model, in view ofstudying its ability to induce polyclonal antibodies that neutralizenative TNFα in an autologous system.

Example 4 Study of the Acute Toxicity of the KLH-TNFα (Human) Kinoid inMice

The goal of the study was to define the characteristic properties of avaccine composition comprising a KLH-TNFα (human) kinoid according tothe invention, to be used as a broad spectrum therapeutic vaccine foruse in the treatment of cancerous cachexia and/or various auto-immunediseases (e.g. rheumatoid arthritis, Crohn's disease and psoriasis).

Particularly, the vaccine composition properties as regards toleranceand absence of risk have been studied.

Consequently, a first toxicity study has been performed in SWISS mice.

A. Material and Methods

Forty six (46) female SWISS mice have been distributed in (i) one groupof four non-treated animals and (ii) three groups of fourteen animalsthat have received, respectively:

a) Control Group (Adjuvant Vehicle): n=14

An intramuscular route injection (IM) located in the thigh of phosphatebuffer saline (PBS);

The control group was used to assess the local and systemic reactivityof the adjuvant use in the formulation.

b) Treated Group (First Dose: 2,000 Times the Single Therapeutic HumanDose, STHD°/N+14.

An intramuscular route injection (IM) located in the thigh of KLH-TNFα(human) kinoid at 50 μg/mouse in a phosphate buffer saline (PBS);

c) Treated Group (Second Dose: 4000 Times the Single Therapeutic HumanDose, STHD): n=14

An intramuscular root injection (IN) located in the thigh of theKLH-TNFα (human) kinoid at the dose of 100 μg/mouse in a phosphatebuffer saline (PBS).

d) Group of Naïve Mice, Non-Treated, that were Included in this Study(n=4).

The computation of the dose concerns the single dose for human use,which is 80 μg/injection/individual: 1 SHTD (single therapeutic humandose), thus, 1.15 μg/kg.

The injections of the KLH-TNFα (human) kinoid have been performed at Day5 (D 5) of the experimentation.

The response of animals to the treatment above has been tested accordingto the following parameters:

a—The eventual number of death, immediate, short time period and longtime period (observation period of 10 days after administration).

b—Local or systemic reactivity to the treatment.

c—Curve showing the general status and the weight of the animals until10 days following administration of the KLH-TNFα (human) kinoid (D 15).

Five (5) days after the observation period (D20), the surviving animalshave been sacrificed and the following controls have been performed.

d—Microscopic anatomo-pathologic examination of the muscle at thelocation of the injection site, for evaluating the local tolerance.

d—Determination of the weight of lungs, heart, liver, and spleen, as anindex value of the organ response to the administration of theimmunostimulating substances.

B. Results

When administered to mice at highly elevated doses (about 4000 times thesingle human therapeutic dose in each mouse), the KLH-TNFα (human)kinoid has not led to any adverse effect, during the whole observationperiod of 10 days, and for each of the tested groups, as it isillustrated by:

1) No occurrence of an immediate or medium term death;

2) No local reaction at the site of injection, nor any systemicreaction;

3) No effect on the growth curves of the mice, in any of the testedgroups.

Further, the macroscopic examination of the organs from the animalssacrificed at the end of the test (D20) has shown no organ alterationnor any increase in the volume from the spleen and the liver.

Further, the weight of the heart, the lungs, the liver and the spleenhave varied in a similar manner for the whole groups of animals tested.

Example 5 Study of the Immunogenicity of the KLH-TNFα (Human) Kinoid ina Transgenic Mice Model Suited for Human TNF

The immunogenic activity (humoral) of the KLH-TNFα kinoid preparation,as compared with the immunogenic activity of the KLH, has been studiedin mice B6.SJL-Tg (TNF) N2 of 5 weeks (group of 10 mice). These micehave been provided by Taconic Company (USA) and consist of mice whichare transgenic for the human TNFα gene (hemizygote).

A. Material and Methods

At Day 0 and at Day 7, mice (group of 10 mice) have received aninjection of 0.2 ml (30 μg) of an emulsion in ISA51 by the intramuscularroot. A second injection of 25 μg in ISA51 was given at Day 28. Then, aretro-orbital blood sampling is performed in each mouse at Day 35.

B. Results

1. Humoral Response

The humoral response is measured by the presence in the sera of theimmunized mice of IgG type antibodies directed against human TNFα; Thehumoral response is determined by an ELISA assay and is expressed at theantibody titer (dilution-1 giving an optical density greater than 0.3).FIGS. 14A-14B illustrate the antibody titer that was obtained.

The sera from mice immunized with the KLH-TNFα (human) final productobtained according to example 35 possess high level of IgG type antibodytiters, whereas the sera from mice immunized with KLH are devoid ofthese antibodies.

The neutralizing activity of these antibodies has been measured with theTNFα cytotoxicity assay on L929 cells. The results are presented inFIGS. 15A-15B.

The antibodies induced by the KLH-TNFα (human) kinoid preparation have ahigh level of neutralizing activity.

Example 6 Study of the Immunogenicity of the KLH-TNFα (Human) Kinoid inthe Rhesus Macaque

The humoral immunogenic activity of the KLH-TNFα (human) kinoid, ascompared with the immunogenic activity of KLH alone, has been studied inrhesus macaque provided by MDS pharma (Lyon-France). It is to be noticedthat the TNFα that is naturally produced by the macaque rhesus shares98.1% amino acid homology with human TNFα.

A—Material and Methods

At Day 0, Day 21 and Day 49, macaques have received an injection of 0.5ml of an emulsion of the kinoid in ISA51 by the intramuscular route,containing (i) either 80 or 20 μg of the KLH-TNFα (human) kinoidpreparation, or (ii) KLH alone. A blood sampling has been performed oneach animal at Day 28, Day 56, and Day 68.

B—Results

1—Humoral Response:

The humoral response has been measured by detecting the presence of IgGisotype antibodies directed against human TNFα in the sera of theimmunized macaques. The humoral response has been determined with anELISA assay and is expressed as antibody titers (dilution-1 giving anoptical density greater than 0.3). The results of the antibody titersobtained are reported in FIGS. 16A-16C.

The sera from the macaques immunized with the KLH-TNFα (human) kinoidpreparation have anti-TNFα IgG isotype antibody titers, whereas serafrom the macaques immunized with KLH alone is devoid of theseantibodies.

The anti-TNFα antibody titers are more important in the serum of themacaques that have received 80 μg of the KLH-TNFα (human) kinoid.

The neutralizing activities of the antibodies have been measured withthe TNFα cytotoxicity assay on L929 cells. The results of these assaysare reported in FIGS. 17A-17C.

The antibodies induced by the KLH-TNFα (human) have a very highneutralizing activity.

Example 7 Assessment of the Therapeutic Efficiency of the ActiveImmunization Against TNFα in huTNFα Transgenic Mice

The assessment of the therapeutic efficiency of the active immunizationstrategy against TNFα using the KLH-TNFα (human) kinoid preparation asthe active ingredient have been performed in transgenic mice huTNFαB6.SJL-Tg (TNF) N2 of 5 weeks old, provided by Taconic Company (USA).These TNFα transgenic mice develop a spontaneous polyarthritis at theage of 4 to 5 weeks.

A—Material and Methods

1—Immunization.

Mice have received one injection of 0.2 ml of an emulsion in ISA51 bythe intramuscular route at days D0, D7, and D28. Four groups of 10 micehave been treated as detailed hereunder:

-   -   Group A: PBS: 200 μl PBS    -   Group B: KLH: 200 μl KLH    -   Group C: KLH-TNF: 200 μl KLH-TNF    -   Group D: KLH-TNF+MTX: 200 μl KLH:TNF and methotrexate (1 mg/kg)        three times a week, starting from immunization No 1 and until        sacrifice (intraperitoneal injection of 200 μl per injection).

Mice have received (i) at Day 0 and Day 7: 30 μg of the KLH-TNFα (human)kinoid preparation and (ii) at Day 28: 15 μg of the same preparation.

A retro-orbital blood sampling was performed on each mouse at Day 35. AtDay 57, at the time the mice are sacrificed, a blood sampling has alsobeen performed.

2—Clinical Examination and Quantitative Assessment of Arthritis:

A clinical examination has been performed, yet at the starting time ofthe experiment, and then twice a week.

The assessments were performed by an observer having no knowledge of thetreatment that was applied. The clinical severity of arthritis at eachjoint (fingers, tarsus, ankle, carpe) was quantified by attribution of ascore varying from 0 to 4 wherein: 0=normal; 1) erythema; 2=swelling;3=deformation; and 4=major deformation or necrosis. A sum of thesescores was performed in order to obtain an arthritis score for eachanimal, every day. A mean for each group was calculated for every day oftreatment.

3—Histological Examination and Quantitative Evaluation of the Arthritis.

The animals were all sacrificed 57 days after the starting of theexperiment. The posterior paws has been removed, fixed in formol,decalcified, then dehydrated and then included in paraffin blocks. Then,5 μm thick histological slices were performed with a microtome. At leastfor serial sections were performed for each paw in order to ensure acorrect spatial assessment of the joint affections. The slidespreparations were then stained by hematoxylin and eosin and thenobserved under optical microscope. The lesions were quantitativelyassessed on each section according to a three points scale (0=normal;3=severe). This histological score may be divided into two parameters:destruction of the cartilage and of the bone (thickness of the cartilageand of the bone, irregularities and presence of erosions) on one hand,and on the other hand, inflammation (synovial proliferation, cellinflammatory infiltration).

4—Statistics:

The results values are given as mean and standard deviation from themean (SDM). A student's t test as well as a variance analysis (ANOVA)have been performed.

B—Results:

1—Humoral Response:

The humoral response is measured by detecting the presence of IgGisotype antibodies directed against human TNFα in the sera of theimmunized macaques. The humoral response is determined by an ELISA assayand the results are expressed as the antibody titer (dilution-1 givingan optical density greater than 0.3).

The results of the antibody titers that were obtained are reported inFIGS. 18A-18D.

2—Clinical Examination and Quantitative Assessment of the Arthritis.Evolution of the Clinical Score with Time.

The evolution of the clinical score with time is reported in FIG. 19.

Treatment of the macaques (i) with KLH-TNF (human) kinoid or (ii) withKLH-TNF (human) kinoid combined with MTX induces a major statisticallysignificant decrease of the arthritis scores that were assessed byclinical examination. By comparison, the arthritis scores determined forthe control animals treated with KLH or with PBS buffer were far lower.

Assessment of the Disease Occurrence and Severity Occurrence Parameters

The assessment of the disease occurrence and disease severity isreported in table 4 hereunder.

-   -   The day of occurrence of the disease has been determined, for        each animal, through a clinical examination. The animals that        had never developed the disease were not taken into account    -   The score “A MAX” corresponds to the maximal score reached by        each animal during the experiment. The score “A MAX” represents        a parameter of disease severity.    -   The incidence means the number of animals having developed        arthritis before the end oft the experiment, it being taken into        account the total number of animals in each group.

TABLE 17 Day of disease occurrence (sick Amax Treatment animals only)Scores ± sted dev. Incidence PBS 24.4 ± 2.5     11.5 ± 4.2  10/10 KLH25.9 ± 2.5     9.0 ± 1.4 10/10 KLH/TNF   45 ± 2.3**/##     0.9 ± 0.5**/# 3/10 KLH/TNF 43.5 ± 3.3*/##      1.0 ± 0.32**/#  6/10 *p < 0.01 vs KLH**p < 0.001 vs KLH #p < 0.02 vs PBS ##p < 0.001 vs PBS (test t ofStudent)

The results show that the treatment by KLH-TNFα and by KLH-TNFα+MTXinduces, in a statistically significant manner:

-   -   a delay in the occurrence of the arthritis, as compared with the        control animals treated by KLH alone or by PBS.    -   a decrease in the severity of the arthritis; and    -   a decrease in the number of sick animals.

3—Histological Examination and Quantitative Assessment of the Arthritis.

The results are reported in table 18 hereunder.

TABLE 18 Inflammation Destruction Treatment Scores ± sem Scores ± semPBS 1.30 ± 0.09 0.71 ± 0.11 KLH 1.53 ± 0.21 1.10 ± 0.23 KLH/TNF   0.16 ±0,.09**   0.08 ± 0,.03** KLH/TNF  0.24 ± 0.09**  0.21 ± 0.12* *p < 0.01vs KLH et vs PBS **p < 0.001 vs KLH et vs PBS (Student's t test)

Treatment with KLH-TNF and with KLH-TNF+MTX induces, in a statisticallysignificant manner, a decrease in the histological alterations(destruction and joint inflammation parameter).

C—Conclusion

Vaccination of the huTNFa transgenic mice with KLH-TNFα (human) kinoidpreparation according to example 1 clearly protects the animals frominflammation and joint destruction, as it is shown by the results of theclinical and histological analysis.

The groups of animals treated with KLH-TNFα (human) kinoid preparation,as regards the joint protection, cannot be distinguished from the groupsof animals treated with KLH-TNFα (human) kinoid preparation combinedwith MTX; These results may be explained by the fact that the majorefficiency of the KLH-TNFα (human) kinoid alone masks, in theseexperimental conditions, the eventual beneficial effect of MTX.

The groups of animals treated by KLH cannot be distinguished from thegroups of animals treated by PBS buffer.

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Modifications and variations of the above-described embodiments of thepresent invention are possible, as appreciated by those skilled in theart in light of the above teachings. It is therefore to be understoodthat, within the scope of the appended claims and their equivalents, theinvention may be practiced otherwise than as specifically described.

1. A method for preparing a stable immunogenic product comprisingantigenic heterocomplexes of TNFα and a Keyhole Limpet Haemocyanin(KLH)carrier protein, comprising the steps of: a) providing a liquid solutioncontaining TNFα; b1) adding one or more antioxidant compounds to saidliquid solution containing TNFα of step a); said antioxidant compoundbeing selected from the group consisting of EDTA, acetyl cysteine,ascorbic acid, ascorbyl glucoside, calcium ascorbate, sodium ascorbate,disodium ascorbyl sulfate, magnesium ascorbate, magnesium ascorbylphosphate, caffeic acid, and cysteine; b2) adding, to the liquidsolution obtained at the end of step b1), one or more compounds thatinduce exposition to the solvent of the hydrophobic portions of TNFα; c)adding a carrier protein to the liquid solution obtained at the end ofstep b), so as to obtain a liquid measure of TNFα and said carrierprotein; said carrier protein consisting of KLH; d) addingglutaraldehyde to the liquid mixture obtained at the end of step c), soas to partially covalently conjugate TNFα molecules to said carrierprotein and obtain heterocomplexes between TNFα and said carrierprotein; e) removing glutaraldehyde and free molecules of both TNFα andsaid carrier protein from the solution obtained at the end of step d),so as to obtain a liquid solution containing purified heterocomplexesbetween TNFα and said carrier protein; f) adding formaldehyde to theliquid solution obtained at the end of step e),and maintaining thepresence of formaldehyde for a period of time ranging from 48 hours to240 hours; g) adding glycine to the heterocomplexes between TNFα andsaid carrier protein obtained at the end of step f); and h) removingformaldehyde and glycine from the liquid solution obtained at the end ofstep g), so as to obtain a liquid solution containing stabilizedheterocomplexes between TNFα and said carrier protein.
 2. The method ofclaim 1, wherein step b2) comprises adding to the liquid solutionobtained at the end of step b1) a compound that induces exposition tothe solvent of the hydrophobic portions of TNFα and wherein saidcompound consists of DMSO.
 3. The method of claim 1, wherein step d)comprises the following steps: d1) adding glutaraldehyde to the liquidmixture obtained at the end of step c), so as to partially covalentlyconjugate TNFα molecules to said carrier protein and obtainheterocomplexes between TNFα and said carrier protein; and d2) addingone or more antioxidant compounds to the heterocomplexes between TNFαand said carrier obtained at the end of step d1), said antioxidantcompound being selected from the group consisting of EDTA, acetylcysteine, ascorbic acid, ascorbyl glucoside, calcium ascorbate, sodiumascorbate, disodium ascorbyl sulfate, magnesium ascorbate, magnesiumascorbyl phosphate, caffeic acid, and cysteine.
 4. The method of claim1, wherein at step a) TNFα concentration ranges from 0.1 mg/mL to 50mg/mL.
 5. The method of claim 1, wherein at step a) TNFα concentrationranges from 0.5 mg/mL to 10 mg/mL.
 6. The method of claim 1, wherein atstep b), the antioxidant consists of EDTA.
 7. The method of claim 6,wherein the final EDTA concentration ranges from 1 mM to 10 mM.
 8. Themethod of claim 2, wherein at step b2) the final DMSO concentrationranges from 5% v/v to 20% v/v.
 9. The method of claim 1, wherein at stepc) the molar ratio of TNFα to said carrier protein ranges from 5: 1 to100:1.
 10. The method of claim 1, wherein at step d), the finalglutaraldehyde concentration ranges from 0.05% w/w to 0.5% w/w.
 11. Themethod of claim 3, wherein at step d2), when the antioxidant consists ofEDTA, then the final EDTA concentration ranges from 1 mM to 10 mM. 12.The method of claim 1, wherein at step e) glutaraldehyde is removed byperforming a dialysis or by performing an ultrafiltration withdiafiltration.
 13. The method of claim 1, wherein at step f), the finalconcentration of formaldehyde ranges from 1% w/w to 10% w/w.
 14. Themethod of claim 1, wherein at step f), the final concentration offormaldehyde ranges from 2% w/w to 5% w/w.
 15. The method of claim 1,wherein at step f) the presence of formaldehyde is maintained during aperiod of time ranging from 96 hours to 192 hours.
 16. The method ofclaim 1, wherein at step f) the presence of formaldehyde is maintainedduring a period of time ranging from 120 hours to 168 hours.
 17. Themethod of claim 1, wherein at step g) the final glycine concentrationranges from 0.01 M to 10 M.
 18. The method of claim 1, wherein at stepg) the final glycine concentration ranges from 0.05 M to 2 M.
 19. Themethod of claim 1, wherein at step h) formaldehyde and glycine areremoved by performing a dialysis, but performing an ultrafiltration withdiafiltration, or by performing Tangential Flow Filtration (TFF).
 20. Amethod of preparing a vaccine composition comprising the steps of: a)preparing a stable immunogenic product comprising antigenicheterocomplexes of TNFα by performing the method of claim 1; and b)combining said stable immunogenic product comprising antigenicheterocomplexes of TNFα prepared at step a) with one or more oilemulsion-based or emulsifier-based immunoadjuvants.